WO2011158883A1 - Antibody capable of determination of cancer prognosis, and cancer prognosis determination method - Google Patents

Antibody capable of determination of cancer prognosis, and cancer prognosis determination method Download PDF

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Publication number
WO2011158883A1
WO2011158883A1 PCT/JP2011/063746 JP2011063746W WO2011158883A1 WO 2011158883 A1 WO2011158883 A1 WO 2011158883A1 JP 2011063746 W JP2011063746 W JP 2011063746W WO 2011158883 A1 WO2011158883 A1 WO 2011158883A1
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cancer
human
antibody
protein
prognosis
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PCT/JP2011/063746
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French (fr)
Japanese (ja)
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浩文 山本
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国立大学法人大阪大学
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Priority to JP2012520483A priority Critical patent/JP5881604B2/en
Publication of WO2011158883A1 publication Critical patent/WO2011158883A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C07K16/3046—Stomach, Intestines
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/56—Staging of a disease; Further complications associated with the disease

Definitions

  • the present invention mainly relates to an antibody that can be used for easily and reliably determining the prognosis of a cancer patient, and a method, kit, and diagnostic agent for simply and reliably determining the prognosis of a cancer patient.
  • Cancer is one of the leading causes of human death worldwide. Despite recent technological advances, particularly in the field of chemotherapy, the number of deaths from cancer is very high. For example, the total death toll from colorectal and rectal cancer is estimated to be about 49,920 in 2009.
  • Infiltrating cells are known as one of risk factors for cancer recurrence and metastasis.
  • a method for detecting only infiltrating cells, particularly active infiltrating cells that can cause hematogenous metastasis in cancer, particularly colorectal cancer has not been known.
  • lymph node metastasis has been widely used as a prognostic indicator in patients with colorectal cancer, especially after surgery.
  • cancer recurrence may occur even if the colon cancer patient after surgery is negative for lymph node metastasis due to pathological findings.
  • cancer recurrence may not occur even if the lymph node metastasis is positive.
  • C4.4A protein which is a glycosylphosphatidylinositol (GPI) -anchored membrane protein, was initially expressed in rat-derived highly metastatic pancreatic cancer cells and was found not to be expressed in normal cancer cells. It was done.
  • GPI glycosylphosphatidylinositol
  • Non-Patent Documents 2 to 4 the expression of the human C4.4A gene has been found in some human malignant tumors. Furthermore, there is a clinical result that the expression of the human C4.4A gene transcript is correlated with poor prognosis in patients with non-small cell lung cancer (see Non-Patent Document 5). However, it is not known at all how the human C4.4A protein can be used to easily and reliably determine the prognosis of cancer.
  • an object of the present invention is to solve the above-described problems of the conventional art. Specifically, antibodies that can be used to easily and reliably determine the prognosis of cancer patients by specifically detecting cancer infiltrating cells, and methods for easily and reliably determining the prognosis of cancer patients
  • the main objective is to provide kits and diagnostics.
  • an antibody that recognizes the amino terminal (N-terminal) site of human C4.4A protein can detect the expression of human C4.4A protein in cancer cells.
  • an antibody that recognizes a specific sequence at the carboxyl terminal (C-terminal) site of human C4.4A protein can detect the expression of human C4.4A protein in cancer cells.
  • using an antibody that recognizes a specific sequence at the C-terminal site of the human C4.4A protein it is possible to detect the state in which the human C4.4A protein is specifically expressed in cancer infiltrating cells, and It was found that the expression pattern of human C4.4A protein is highly correlated with the prognosis of cancer patients.
  • the present invention has been completed by making further improvements based on such findings.
  • Item 1 An anti-human C4.4A protein antibody that specifically binds to a region of the amino acid sequence shown in SEQ ID NO: 1.
  • Item 2. The antibody according to Item 1, which is a polyclonal antibody.
  • Item 3. The antibody according to Item 1, which is a monoclonal antibody.
  • Item 6. The monoclonal antibody according to Item 3, which is a monoclonal antibody produced by a hybridoma specified by NITE BP-1090.
  • Item 5-1 An anti-human C4.4A protein antibody that specifically binds to a region of the amino acid sequence shown in SEQ ID NO: 1.
  • Item 2. The antibody according to Item 1, which is a polyclonal antibody.
  • Item 3. The antibody according to Item 1, which is a monoclonal antibody.
  • Item 6. The monoclonal antibody according to Item 3, which is a monoclonal antibody produced by a hybridoma specified by NITE BP-1090.
  • Item 5-1 An anti-human C
  • a method for determining the prognosis of a cancer patient (1) a step of detecting expression of a human C4.4A protein with the antibody according to any one of Items 1 to 4 in a cancer tissue collected from a cancer patient; (2) Based on the detection result of the step (1), the localization of the human C4.4A protein is compared between the cell membrane and the cytoplasm in the cells of the advanced cancer invasion, and the advanced cancer invasion and the cancer lesion are compared.
  • the human C4.4A protein is Prognosis when the expression level of the human C4.4A protein is accumulated on the cell membrane of some cells and is increased in the cells in the cancer invasion advanced portion as compared with the surface layer portion or middle layer portion of the cancer lesion. Determining a failure. Item 5-2.
  • a method for determining the prognosis of a cancer patient (1) performing immunostaining using the antibody according to any one of Items 1 to 4 in a cancer tissue collected from a cancer patient; (2) Based on the immunostaining in the above step (1), the staining intensity of human C4.4A protein is compared between the cell membrane and the cytoplasm in the cells of the advanced cancer invasion, and the advanced cancer invasion and the cancer lesion are compared. Comparing the staining intensity of the human C4.4A protein with the surface layer portion or the middle layer portion of the above, and (3) staining of the human C4.4A protein based on the comparison of the staining intensity of the step (2).
  • Intensity is enhanced in the cell membrane of the advanced cancer invasion cell compared to the cytoplasm of the advanced cancer invasion cell, and the staining intensity of the human C4.4A protein is increased in the advanced cancer invasion cell.
  • a method for determining the prognosis of a cancer patient (1) A step of immunoassaying a cancer tissue collected from a cancer patient with respect to the human C4.4A protein with the antibody, (2) About the result of the immunoassay in the step (1), a step of comparing the measurement result between the advanced portion of cancer invasion and the surface layer portion or middle layer portion of the cancer lesion, and (3) of the step (2) Based on the comparison of the measurement results, the human C4.4A protein is accumulated on the cell membrane of the cancer invasion advanced portion cell, and the expression level of the human C4.4A protein is increased in the cancer invasion advanced portion cell. And a step of determining that the prognosis is poor when cells are elevated compared to cells in the surface layer or middle layer of the lesion. Item 5-4.
  • a method for determining the risk of cancer recurrence in a cancer patient (1) a step of detecting expression of a human C4.4A protein with the antibody according to any one of Items 1 to 4 in a cancer tissue collected from a cancer patient; (2) Based on the detection result of the step (1), the localization of the human C4.4A protein is compared between the cell membrane and the cytoplasm in the cells of the advanced cancer invasion, and the advanced cancer invasion and the cancer lesion are compared.
  • the human C4.4A protein is Prognosis when the expression level of the human C4.4A protein is accumulated on the cell membrane of some cells and is increased in the cells in the cancer invasion advanced portion as compared with the surface layer portion or middle layer portion of the cancer lesion. Determining a failure.
  • Item 6. The method according to any one of Items 5-1, 5-2, or 5-3, wherein the prognosis is a prognosis based on a risk of cancer recurrence.
  • Item 7. Item 7.
  • Item 5-4 or Item 6 wherein the cancer recurrence is cancer recurrence due to hematogenous metastasis.
  • Item 8-1 Item 8. The method according to any one of Items 5-1, 5-2, 5-3, 5-4, 6 or 7, wherein the cancer patient is a patient with cancer of the digestive system.
  • Item 8-2 Item 8. The method according to any one of Items 5-1, 5-2, 5-3, 5-4, 6 or 7, wherein the cancer patient is a colorectal cancer patient.
  • Item 9-1 A kit for determining the prognosis of a cancer patient, comprising the antibody according to any one of Items 1 to 4. Item 9-2.
  • a kit for determining the risk of cancer recurrence in a cancer patient comprising the antibody according to any one of Items 1 to 4.
  • Item 10. Item 9. The kit according to Item 9-1, wherein the prognosis is a prognosis based on the risk of cancer recurrence.
  • Item 11. Item 10. The kit according to Item 9.2 or Item 10, wherein the cancer recurrence is cancer recurrence due to hematogenous metastasis.
  • Item 12-1 The kit according to any one of Items 9-1, 9-2, 10 or 11, wherein the cancer patient is a patient with cancer of the digestive system. Item 12-2. Item 12.
  • a diagnostic reagent for cancer recurrence risk comprising the antibody according to any one of Items 1 to 4.
  • a cancer diagnostic reagent comprising the antibody according to any one of Items 1 to 4.
  • Item 14. Item 13.
  • the diagnostic reagent according to Item 13-1 or Item 13-2, wherein the cancer recurrence is cancer recurrence due to hematogenous metastasis.
  • the antibody of the present invention can detect human C4.4A protein specifically expressed in cancer infiltrating cells.
  • the determination method can be easily performed using the kit or the cancer diagnostic reagent of the present invention.
  • the present invention it is possible to determine with high accuracy whether or not necessary aftercare such as administration of an anticancer drug is necessary particularly in patients with colorectal cancer and the like. As a result, it is expected that the treatment that burdens the patient will be suppressed, leading to a reduction in social security costs.
  • FIG. 1 shows the amino acid sequence of the human C4.4A protein and the two sequences (bold) used as antigens.
  • the GPI anchor addition signal sequence is shown enclosed in a box.
  • FIG. 2 shows human C4.
  • HCT116, KM12SM colon cancer-derived cell lines
  • N normal specimens
  • T colon cancer tissue specimens
  • FIG. 3 shows anti-C4.4A-1 polyclonal antibody (A, C) and anti-C4.4A-2 polyclonal antibody for normal esophageal mucosa tissue sections (A, B) and colon cancer tissue sections (C, D). The result of immunohistochemistry using (B, D) is shown.
  • A, B Staining was observed in the squamous epithelium on the base.
  • C No signal was detected in the anti-C4.4A-1 polyclonal antibody in the colon cancer tissue specimen.
  • D A staining signal was obtained with the anti-C4.4A-2 antibody in the same sample. Magnification: A, B: x100; C, D: x40.
  • FIG. 4 shows the results of immunohistochemical analysis of human C4.4A protein expression using anti-C4.4A-2 polyclonal antibody.
  • A A specific expression pattern of human C4.4A protein in a cancer invasion advanced part and an invasion site of a colon cancer tissue specimen is shown. In the surface layer or middle layer of the cancer lesion, weak expression of human C4.4A protein is detected in the cytoplasm in most cases, while in the cancer invasion advanced portion, the expression of C4.4A protein is frequently detected on the cell membrane. It was done. Of the enormous number of cancer tissue specimens, only the cells facing the neoplastic stroma expressed human C4.4A protein (lower left panel). Magnification: upper left: x12.5; lower left x40; upper right, lower right: x100.
  • (B) shows the result of immunostaining in tumor exudation.
  • the exudate cells have fallen from the cancer invasion advanced part of the colon cancer tissue. This was classified as a type A expression pattern. Magnification: x100.
  • (C) A significant correlation was found between the frequency of sputum and the expression pattern of human C4.4A protein in advanced cancer invasion (P ⁇ 0.0001).
  • FIG. 5 shows the results of immunohistochemistry with anti-C4.4A-2 polyclonal antibody at the lymph node metastasis site and the lung infiltration site.
  • Lymph node metastasis site Infiltrating tumor cells that infiltrate the fibrous cap were specifically expressed with strong human C4.4A protein on the cell membrane.
  • Lung infiltration site Infiltrating tumor cells in the periphery of the infiltration (arrow site) showed a similar expression pattern. Magnification: upper left: ⁇ 12.5; lower left: ⁇ 40; upper right, lower right: ⁇ 100.
  • FIG. 6 shows the relationship between the expression pattern of human C4.4A protein and the prognosis of patients (5-year overall survival rate).
  • FIG. 7 shows the relationship between the expression pattern of human C4.4A protein and the prognosis of patients (5-year disease-free survival rate).
  • FIG. 8 shows the amino acid sequence of human C4.4A protein and the two sequences (bold) used as antigens.
  • the GPI anchor addition signal sequence is shown enclosed in a box.
  • FIG. 9 shows the use of anti-C4.4A-2 polyclonal antibody, anti-C4.4A-2-2 polyclonal antibody and anti-C4.4A-2-3 polyclonal antibody in serial sections of colorectal cancer tissue specimen (advanced advanced part). The results of immunohistochemical analysis were shown. Magnification: x10.
  • FIG. 10 shows anti-C4.4A-2 polyclonal antibody (in the figure) in serial sections of the invasion advanced part (Invasive front) (right column and middle column) and middle layer part (Intermediate portion) (left column) of colorectal cancer tissue specimens.
  • C4.4A-2 upper panel
  • anti-C4.4A 119
  • polyclonal antibody in the figure, C4.4A (119), middle panel
  • anti-C4.4A (277) polyclonal antibody in the figure, C4.4A
  • (119), lower panel) shows the results of immunohistochemical analysis. Magnification: x10.
  • FIGS. 11A and B show immunohistochemistry using anti-C4.4A-2 polyclonal antibody (left, polyclonal) and anti-C4.4A-2 monoclonal antibody (right, monoclonal) in a colon cancer tissue specimen (advanced advanced part). The result of the analysis is shown.
  • anti-C4.4A-2 monoclonal antibody was used, the expression of C4.4A protein was detected on the cell membrane at the advanced cancer invasion portion, as with the anti-C4.4A-2 polyclonal antibody. Magnification: x20.
  • FIGS. 12A and B show anti-C4.4A-2 monoclonal antibody (left side, mAb) and anti-C4.4A-2 monoclonal antibody (right side, mAb) pre-treated with excess target peptide in serial sections of colon cancer tissue specimens. The results of immunohistochemical analysis using (+ peptide) are shown. A and B show the results using different serial slice samples. No staining was obtained when anti-C4.4A-2 monoclonal antibody pretreated with an excess amount of target peptide was used. Magnification: x20.
  • FIG. 13 shows the results of Western blot analysis.
  • Anti-C4.4A-2 polyclonal antibody Polyclonal, Ab
  • Anti-C4.4A-2 monoclonal antibody Monoclonal, Ab
  • Anti-C4.4A-2 monoclonal antibody Monoclonal, Ab
  • a band a band indicated by an arrow in the figure
  • Detection of actin is a loading control.
  • the band indicated by * is a band detected nonspecifically.
  • the main object of the present invention is to provide antibodies, methods, and kits for easily and reliably determining the prognosis of cancer patients.
  • the antibody, method, kit, and diagnostic agent will be described in this order.
  • Antibody The antibody of the present invention is an antibody that specifically binds to the region of the amino acid sequence shown in SEQ ID NO: 1.
  • the amino acid sequence shown in SEQ ID NO: 1 is the 301st to 316th region of the full-length human C4.4A protein shown in SEQ ID NO: 3.
  • the human C4.4A protein is a protein having the amino acid sequence shown in SEQ ID NO: 3, or an amino acid sequence of a part of SEQ ID NO: 3 containing at least the first to eighth amino acid sequences of the amino acid sequence shown in SEQ ID NO: 1. Any protein that has
  • the specific binding of this antibody to the region of the amino acid sequence shown in SEQ ID NO: 1 can be visualized by a known method.
  • immunoassay methods for example, ELISA method
  • immunoelectrophoresis methods for example, Western blot method
  • immunohistochemistry for tissues or cells.
  • the antibody of the present invention is a fluorescent substance (for example, fluorescein isothiocyanate), a radioisotope (for example, iodine 125), an enzyme (for example, alkaline phosphatase, horseradish peroxidase), and other proteins (for example, avidin). It may be labeled with a molecule such as Alternatively, a secondary antibody that specifically recognizes the antibody of the present invention may be used.
  • the antibody of the present invention is not limited in its immunoglobulin class as long as it can specifically bind to the amino acid sequence region shown in SEQ ID NO: 1.
  • IgG, IgM, IgA, IgD, IgE, IgY and the like can be used. Can be mentioned.
  • the subclass is not limited. Preferably, it is IgG.
  • the antibody of the present invention is derived from a mammal (for example, human, mouse, rat, rabbit) or bird (for example, chicken or ostrich) capable of producing the antibody.
  • a mammal for example, human, mouse, rat, rabbit
  • bird for example, chicken or ostrich
  • the antibody of the present invention may be a polyclonal antibody or a monoclonal antibody.
  • a chimeric antibody, a humanized antibody, or a human antibody may be used among monoclonal antibodies.
  • the chimeric antibody and the humanized antibody refer to an antibody derived from a human other than a site capable of binding to an antigen among immunoglobulin proteins.
  • a human antibody refers to a human-derived antibody or an antibody derived from a human antibody-producing mouse having a complete human-derived immunoglobulin gene.
  • Chimeric antibodies, humanized antibodies, and human antibodies have low immunogenicity in humans and are useful in using antibodies in medical preparations and the like. Therefore, when the antibody of the present invention is used for medical preparations, monoclonal antibodies are preferable, and humanized antibodies or human antibodies are particularly preferable.
  • the antibody of the present invention may be a full-length immunoglobulin protein or a fragment thereof containing a site capable of binding to an antigen. It may also be a composition containing an antibody, such as antiserum.
  • the antibody of the present invention can be prepared by administering an antigen to an immunized animal.
  • an antigen a peptide comprising the amino acid sequence shown in SEQ ID NO: 1 can be used.
  • the peptide has a small molecular weight and may not cause an immune response sufficiently. Therefore, the peptide is bound to an appropriate protein (eg, albumin, thyroglobulin) and used as an antigen. May be.
  • the antigen is thyroglobulin added with the amino acid sequence shown in SEQ ID NO: 1.
  • the antibody of the present invention can also be obtained by searching for an antibody that binds to an antigen or a fragment thereof from a library such as a cell or phage that can produce various antibodies or fragments thereof.
  • a peptide comprising the full length of the amino acid sequence shown in SEQ ID NO: 1 or a part of 6 amino acids or more can be used.
  • it is a peptide having the amino acid sequence shown in SEQ ID NO: 1.
  • the method for obtaining a peptide used as an antigen is not particularly limited, and can be a known method.
  • it may be naturally derived, chemically synthesized, cell-free synthesized or recombinant peptide.
  • polyclonal antibody In producing a polyclonal antibody, an immunized animal (for example, mouse, rat, rabbit, chicken) is emulsified in a usual manner, for example, an immunogen solution is emulsified with an equal volume of Freund's complete or incomplete adjuvant. The mixture can be inoculated (primary immunization) and then immunized several times at intervals of 2 to 4 weeks. Thereafter, blood is collected from the immunized animal, and antiserum containing a polyclonal antibody is prepared. Thereafter, the polyclonal antibody can be purified from the antiserum by DEAE ion exchange chromatography or protein G affinity chromatography.
  • a monoclonal antibody can be prepared by isolating a hybridoma (monoclonal antibody-producing cell) that produces a monoclonal antibody.
  • a hybridoma monoclonal antibody-producing cell
  • it can also be prepared by a phage display method, that is, a method of searching for an antibody or antibody fragment that specifically binds to an antigen from a phage library that expresses various antibodies or antibody fragments.
  • the method for separating hybridomas is described in Kohler et al., Nature, 256: 495 (1975), etc., but is briefly described below.
  • the spleen is aseptically removed from the immunized animal (eg, mouse) several days after the final immunization, and spleen cells are prepared from the spleen.
  • Spleen cells are used in the cell fusion step together with myeloma cells (myeloma cells).
  • myeloma cells myeloma cells.
  • myeloma cells myeloma cells.
  • NS-1, P3U1, SP2 / 0, etc. can be used as myeloma cells.
  • spleen cells and myeloma cells in a medium containing a fusion promoter can be used.
  • a conventional mixing ratio ratio of about 1/5 to 1/10.
  • the hybridoma secreting the target monoclonal antibody in the culture supernatant is screened, for example, by the enzyme-linked immunoassay (ELISA) for the reactivity between the culture supernatant and the peptide having the amino acid sequence of SEQ ID NO: 1. You can confirm and choose by doing.
  • ELISA enzyme-linked immunoassay
  • Monoclonal antibodies produced by hybridomas can be easily prepared by culturing them.
  • the culture can be performed in an appropriate medium (for example, Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum) or in vivo (for example, in the peritoneal cavity of a mouse).
  • DMEM Dulbecco's modified Eagle medium
  • the antibody can also be purified using the culture supernatant or ascites as a starting material.
  • a general method for protein purification such as ammonium sulfate salting-out, ion exchange chromatography, gel filtration chromatography, affinity chromatography using protein A or protein G binding polymer, or dialysis, is used. They can be used in appropriate combinations.
  • the phage display method is known and described in Clackson et al., Nature 352: 624-8 (1991), but is briefly described below. From a phage library that displays antigen recognition sites of various antibodies prepared in advance, a phage that displays an antigen recognition site that binds to a peptide having the amino acid sequence shown in Sequence Listing 1 is selected.
  • the antigen recognition site displayed by the selected phage may be used as it is, or a full-length antibody can be obtained by incorporating the full-length antibody into a CHO cell capable of producing the full-length antibody by a genetic engineering technique.
  • Humanized antibody human antibody
  • a humanized antibody is an antibody protein whose origin is other than the minimum necessary region for antigen recognition (complementarity-determining region: CDR). is there.
  • a method for producing a humanized antibody is described in, for example, EP0239400, US5585089, and the like.
  • a human antibody can be obtained from a human antibody-producing mouse having a complete human-derived immunoglobulin gene. Human antibody-producing mice are known and described in WO 97/07671.
  • a preferred example is a monoclonal antibody produced by the hybridoma obtained in the examples described later (indication for identification given by the trustee: CA4.4A Hybridoma 11A1).
  • the hybridoma was commissioned on April 27, 2011 (original deposit date) to the Patent Microbiology Depositary Center, National Institute of Product Evaluation Technology, which has an address in 2-5-8, Kazusa Kamashita, Kisarazu City, Chiba Prefecture, Japan.
  • the hybridoma received a request for transfer from the original deposit to the deposit under the Budapest Treaty on May 31, 2011.
  • the accession number is NITE BP-1090.
  • a preferred example includes a monoclonal antibody produced by a hybridoma specified by the accession number: NITE BP-1090, but is not limited thereto.
  • the hybridoma is a hybridoma that produces an IgG subtype monoclonal antibody prepared from spleen cells of a mouse immunized with a polypeptide having the amino acid sequence shown in SEQ ID NO: 1.
  • the antibody of the present invention specifically binds to the amino acid sequence region shown in SEQ ID NO: 1 and can specifically detect human C4.4A protein.
  • the prognosis of cancer patients for example, prognosis based on the risk of cancer recurrence, particularly the prognosis based on the risk of cancer recurrence due to hematogenous metastasis
  • pharmaceutical preparations eg, cancer recurrence risk
  • diagnostic agents particularly diagnostic agents for cancer recurrence risk due to hematogenous metastasis.
  • the prognosis determination for cancer patients is described in detail below.
  • the present invention also provides the use of the antibody for determining the prognosis of cancer patients and the use of the antibody for the production of a pharmaceutical preparation.
  • the present invention provides a method for easily and reliably determining the prognosis of a cancer patient.
  • the prognosis of a cancer patient to be determined is a prognosis based on cancer recurrence, particularly a prognosis based on cancer recurrence due to hematogenous metastasis.
  • the present invention is also a method for determining the risk of cancer recurrence, particularly the risk of cancer recurrence due to hematogenous metastasis.
  • the type of cancer affected by the cancer patient targeted by the method of the present invention is not limited, and can be any type of cancer target.
  • solid cancer in the stomach, large intestine, lung, liver, prostate, pancreas, esophagus, bladder, gallbladder / bile duct, breast, uterus, thyroid gland, ovary, etc. is preferred, and digestive system such as stomach, large intestine, esophagus Especially preferred is cancer.
  • Colorectal cancer capable of highly accurate determination is more preferable as a target.
  • Colon cancer refers to any part of the large intestine, such as the cecum, colon (ascending colon, transverse colon, descending colon, sigmoid colon), rectal sigmoid, rectum (upper rectum, lower rectum), and anal canal. Including cancer.
  • stage II and stage III cancer patients based on the TMN classification are particularly preferable from the viewpoint of high prognosis determination.
  • the TMN classification is a known index of cancer progression classification determined by the International Union for Cancer (UICC).
  • UICC International Union for Cancer
  • a person skilled in the art for example, a doctor can diagnose the degree of cancer progression based on the TMN classification of cancer patients.
  • the progression of cancer is stage I based on the size of the primary tumor (Tumor), the presence and number of lymph node metastases (Node), and the presence of metastasis (Metastasis). It is classified into ⁇ IV.
  • the cancer patient targeted by the present invention is preferably a patient who has already undergone a therapeutic treatment such as a surgical operation, but is not limited thereto.
  • the prognosis of cancer patients refers to the prediction of the future state of cancer from a certain point of time, for example, at the time of diagnosis or treatment of cancer, and the risk (possibility) of cancer progression, recurrence, and metastasis.
  • the risk is high, the prognosis is poor.
  • the risk (probability) of cancer progression, recurrence, or metastasis is not high, the prognosis is good.
  • “There is a risk of cancer recurrence” is not particularly limited, but for example, about 1% (when infiltrating to the submucosa) or about 6 in the case of stage I colorectal cancer patients There is a risk of cancer recurrence with a probability greater than the statistically known recurrence rate, such as% (when infiltrating into the muscle layer or later), about 13% for stage II, about 30% for stage III Can be pointed to.
  • the prediction index is not specified, for example, overall survival (OS) and disease-free survival (DFS) are preferable.
  • total survival rate generally means the proportion of the observed cases that can be confirmed to be alive after a specific period from the specific observation start time.
  • Disease-free survival rate generally means the proportion of observed cases that can be confirmed to be alive without recurrence of cancer after a specific period from the start of observation.
  • the determination method of the present invention comprises the following steps (1) to (3): (1) detecting the expression of human C4.4A protein with the antibody in cancer tissue collected from a cancer patient; (2) Based on the detection result of the step (1), the localization of the human C4.4A protein is compared between the cell membrane and the cytoplasm in the cells of the advanced cancer invasion, and the advanced cancer invasion and the cancer lesion are compared.
  • the human C4.4A protein is Prognosis when the expression level of the human C4.4A protein is accumulated on the cell membrane of some cells and is increased in the cells in the cancer invasion advanced portion as compared with the surface layer portion or middle layer portion of the cancer lesion. The process of determining that it is defective.
  • the above steps (1) to (3) can also be described as follows.
  • (1) A step of performing immunostaining using the antibody in a cancer tissue collected from a cancer patient, (2) Based on the immunostaining in the above step (1), the staining intensity of human C4.4A protein is compared between the cell membrane and the cytoplasm in the cells of the advanced cancer invasion, and the advanced cancer invasion and the cancer lesion are compared. Comparing the staining intensity of the human C4.4A protein with the surface layer portion or the middle layer portion of the above, and (3) staining of the human C4.4A protein based on the comparison of the staining intensity of the step (2).
  • Intensity is enhanced in the cell membrane of the advanced cancer invasion cell compared to the cytoplasm of the advanced cancer invasion cell, and the staining intensity of the human C4.4A protein is increased in the advanced cancer invasion cell.
  • a step of immunoassaying a cancer tissue collected from a cancer patient with respect to the human C4.4A protein with the antibody (2) About the result of the immunoassay in the step (1), a step of comparing the measurement result between the advanced portion of cancer invasion and the surface layer portion or middle layer portion of the cancer lesion, and (3) of the step (2) Based on the comparison of the measurement results, the human C4.4A protein is accumulated on the cell membrane of the cancer invasion advanced portion cell, and the expression level of the human C4.4A protein is increased in the cancer invasion advanced portion cell.
  • a step of determining a poor prognosis when the invasion advanced portion and cells in the surface layer or middle layer of the cancer lesion are enhanced.
  • a method for detecting the expression of human C4.4A protein in a cancer tissue collected from a cancer patient, that is, performing immunostaining (immunohistochemistry) using the antibody can be performed using a known method.
  • a tissue specimen when performing immunostaining (immunohistochemistry), a tissue specimen may be fixed. Specifically, the specimen is preferably fixed with a formalin solution and dehydrated with an ethanol solution, but is not limited thereto. Furthermore, the immobilized specimen can be embedded in paraffin, and finally a sample section can be prepared from the paraffin block.
  • the specimen and the antibody are brought into contact, and this method is also known.
  • the sample may be brought into contact with a PBS solution containing an antibody at 0.1 to 100 mg / ml and reacted at a temperature of 4 to 37 ° C. for 60 minutes to overnight.
  • the binding between the antibody and the human C4.4A protein is visualized, and this method is also known.
  • a labeled secondary antibody In the case of detecting the fluorescence used for the label for visualization, the visualization can be performed by a fluorescence microscope system equipped with a CCD camera. Alternatively, a developed photograph can be obtained.
  • the localization of the human C4.4A protein is compared between the cell membrane and the cytoplasm in the cells of the advanced cancer invasion, and the advanced cancer invasion and the cancer lesion are compared. Comparing the localization and expression level of the human C4.4A protein between the surface layer and the middle layer.
  • step (1) Evaluate the result of step (1). Evaluation is performed about the location in the cell in which human C4.4A protein is detected, and the relative substance amount detected. Specifically, the localization and expression level of human C4.4A protein are compared based on the expression pattern of human C4.4A protein obtained in step (1). In other words, in the step (2), the staining intensity of the human C4.4A protein is compared between the cell membrane and the cytoplasm in the cells in the advanced part of cancer invasion based on the immunostaining in the step (1), and the cancer Compare the staining intensity of human C4.4A protein between the advanced invasion and the surface layer or middle layer of the cancer lesion
  • An evaluation method for comparing the localization and expression level of human C4.4A protein that is, a method for evaluating the staining intensity of human C4.4A protein may be appropriately selected by those skilled in the art.
  • the “advanced portion of cancer invasion” refers to the deepest portion that grows from the surface layer portion to the deep portion of the tissue in which cancer has become cancerous by pathological findings, and refers to the “surface layer portion or middle layer portion (intermediate portion of the cancer lesion). ”)” Refers to the entire region other than the advanced part.
  • the cell membrane and cytoplasm of cells in advanced cancer invasion can be identified by those skilled in the art by conventional means (eg, identification based on morphology).
  • cancer occurs in the surface layer of the organ (eg, epithelium, mucous membrane, etc.).
  • the original cancerous part is the original lesion.
  • the resulting cancer grows (ie, infiltrates) toward the peripheral site while increasing the number of cells.
  • the periphery refers to the submucosal layer, the muscle layer, and the like in order from the surface layer.
  • the cancer invasion advanced portion the deepest portion of the cancer lesion that contacts non-cancerous peripheral sites such as the submucosa and muscle layers.
  • the cancer invasion advanced portion As the cancer further grows, when the site that was previously in contact with the surrounding site is no longer the deepest part, it becomes the “middle layer (intermediate part)”.
  • the “surface layer portion or middle layer portion of the cancer lesion” may be either the surface layer portion or the middle layer portion.
  • the cancer invasion advanced portion and the middle layer are often close in positional relationship. Therefore, from the viewpoint of practicality, it is preferable to make a comparison between the cancer invasion advanced part and the middle part of the cancer lesion.
  • step (1) When the results of step (1) are compared between the cell membrane and the cytoplasm, and between the cancer invasion advanced portion and the surface layer portion or middle layer portion of the cancer lesion in the cells of the cancer invasion advanced portion, the comparison results are as follows. It can be classified into four patterns (see Example 2 and FIG. 4 described later): (A) Human C4.4A protein is accumulated on the cell membrane of cells in advanced cancer invasion, and the expression level of human C4.4A protein is higher than that in the surface layer or middle layer of cancer lesions in cells in advanced cancer invasion Is increasing, (B1) Human C4.4A protein is accumulated on the cell membrane of cells in advanced cancer invasion, but the expression level of human C4.4A protein is between the advanced cancer invasion and the surface layer or middle layer of the cancer lesion.
  • the comparison result can be rephrased as follows.
  • (A) The staining intensity of human C4.4A protein is enhanced in the cell membrane of cells in advanced cancer invasion compared to the cytoplasm of cells in advanced cancer invasion, and the staining intensity of human C4.4A protein is advanced in cancer invasion.
  • B1 Although the staining intensity of human C4.4A protein is enhanced on the cell membrane of cells in advanced cancer invasion compared to the cytoplasm of cells in advanced cancer invasion, the staining intensity of human C4.4A protein is increased.
  • (B2) The staining intensity of the human C4.4A protein is comparable between the advanced part of cancer invasion and the surface layer or middle part of the cancer lesion.
  • the staining intensity of human C4.4A protein is enhanced in the cells in the advanced part of cancer invasion compared to the surface layer or middle layer of the cancer lesion.
  • the staining intensity of human C4.4A protein is not enhanced on the cell membrane of cells in advanced cancer invasion compared to the cytoplasm of cells in advanced cancer invasion, and the staining intensity of human C4.4A protein is It is the same level between the advanced part of cancer invasion and the surface part or middle part of the cancer lesion.
  • Human C4.4A protein is accumulated on the cell membrane of cells in advanced cancer invasion means that the human C4.4A protein is compared with the cytoplasm of cells in advanced cancer invasion in the cell membrane of advanced cancer invasion. Refers to a state in which the expression level of is increased.
  • step (1) is evaluated and the expression patterns of human C4.4A protein are compared.
  • the human C4.4A protein is accumulated on the cell membrane of the cell in the cancer invasion advanced part, and the expression level of the human C4.4A protein is the cancer invasion advanced.
  • the step of determining a poor prognosis is compared with the surface layer or middle layer of the cancer lesion.
  • careful follow-up and intensive preventive measures as aftercare (mainly aftercare after surgery).
  • careful follow-up and focused preventive measures include fluorouracil (5-FU), levofolinate (LV; trade name: Isobolin, Pfizer Inc.), oxaliplatin (L-OHP; trade name: Elplat) / Eloxatin, Yakult Headquarters) and other measures such as administration of anti-cancer agents are included, but are not limited thereto.
  • the determination method of the present invention may be combined with other known or prognosis determination methods for cancers to be found in the future. Examples include, but are not limited to, a combination of histopathological findings of cancer specimens (specifically, the presence or absence of lymph node metastasis), detection of genetic factors in cancer patients, and the like.
  • the method of the present invention is a method for determining the risk of cancer recurrence, the presence or absence of the risk of cancer recurrence is determined.
  • Kit The kit of the present invention is a kit for determining the prognosis of a cancer patient and comprising the above-described antibody.
  • kit of the present invention may contain other components as necessary in addition to the above-described antibodies.
  • Other components may be, for example, reagents or instruments necessary to perform an immunoassay.
  • reagents used for specimen fixation when performing immunohistochemistry for example, formalin, ethanol
  • reagents used for detection for example, secondary antibody
  • instruments to be used for example, slide glass, cover crow
  • a positive control sample, a negative control sample, etc. are mentioned, However It is not restricted to this.
  • the document etc. which wrote down the procedure for performing the said determination method can be included.
  • the kit of the present invention can be prepared by providing the above-mentioned components as necessary according to a conventional method.
  • the usage form of the kit is not particularly limited, but it is preferably used in the above determination method. When used in the above determination method, the determination can be easily performed.
  • the prognosis of a cancer patient to be determined is a prognosis based on the risk of cancer recurrence, particularly a prognosis based on the risk of cancer recurrence due to hematogenous metastasis.
  • the cancer diagnostic agent of the present invention comprises the antibody described above.
  • One preferred embodiment of the cancer diagnostic agent of the present invention is a diagnostic agent for cancer recurrence risk.
  • a particularly preferred embodiment is a diagnostic agent for cancer recurrence risk due to hematogenous metastasis.
  • the diagnostic agent of the present invention can contain other components as long as the antibody is not inactivated and there is no problem in performing the above-described determination method.
  • Specific examples include physiological saline and buffer solution, but are not limited thereto.
  • the diagnostic agent of the present invention can be prepared by providing the above components as necessary according to a conventional method.
  • Example 1 Preparation of polyclonal antibody and specificity test ⁇ I. Experimental materials and methods> 1. Production of polyclonal antibodies Rabbits were immunized with thyroglobulin conjugated with the target peptide.
  • the target peptide is a peptide having the amino acid sequence shown in SEQ ID NO: 2 corresponding to the 83rd to 97th amino acid sequence near the N-terminal of the human C4.4A protein, and the 301st to 316th of the C-terminal of the human C4.4A protein.
  • a peptide having the amino acid sequence shown in SEQ ID NO: 1 containing a part of the GPI addition signal sequence corresponding to the amino acid sequence was used. (FIG. 1).
  • the antiserum collected from each rabbit was passed through a column in which the target peptide was bound to carrier beads, thereby purifying IgG that specifically binds to human C4.4A protein.
  • the obtained antibodies against the N-terminus and C-terminus were named anti-C4.4A-1 antibody and anti-C4.4A-2 antibody, respectively.
  • Cell line Human colon cancer-derived cell line HCT116 was obtained from American Cell Type Collection (Manassas, VA, USA).
  • the human colorectal cancer-derived cell line KM12SM used was distributed from Professor Kanazawa University Cancer Research Institute. These cell lines are in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 units / mL penicillin, and 100 microgram / mL streptomycin, and at a temperature of 37 ° C. and air Culturing was performed in a constant temperature and humidity chamber having a carbon dioxide concentration of 5%.
  • DMEM Dulbecco's modified Eagle's medium
  • FBS fetal bovine serum
  • streptomycin 100 units / mL
  • streptomycin 100 units / mL
  • Immunohistochemistry 4 micromm thick tissue sections were prepared from paraffin embedded blocks. Antigen activation treatment was performed in a 10 mM citrate buffer solution at a temperature of 95 ° C. for 40 minutes, and then antibody staining was performed using a Betastain ABC peroxide kit (Vector Labs, California, USA). The slides were incubated overnight at 4 ° C. in anti-C4.4A-1 antibody or anti-C4.4-2 antibody diluted 1: 200 in 50 ⁇ g / ml antibody stock solution. Non-immune rabbit-derived IgG (Vector Laboratories) was used as a negative control and used in place of the primary antibody to eliminate the possibility of false positive reaction due to non-specific binding of the secondary antibody or IgG.
  • the rabbit-derived anti-human actin antibody used as a loading control was purchased from Sigma-Aldrich (St. Louis, MO, USA).
  • anti-C4.4A-2 antibody that specifically binds to the region of the amino acid sequence shown in SEQ ID NO: 1 was able to specifically detect human C4.4A protein in a sample derived from colorectal cancer.
  • anti-C4.4A-1 antibody could not detect human C4.4A protein.
  • the human C4.4A protein is predicted to be cleaved between the 308th and 309th amino acid residues of the full-length sequence shown in SEQ ID NO: 3 and a GPI anchor is added. Therefore, the antibody of the present invention is presumed to recognize the region of the 301st to 308th amino acid sequence of the full-length sequence shown in SEQ ID NO: 3.
  • Example 2 Detection of human C4.4A protein in colorectal cancer tissue specimens and correlation with survival rate ⁇ I.
  • Experimental method> 1 Detection of human C4.4A protein in colorectal cancer tissue specimens According to the method according to Example 1, detection of human C4.4A protein in colorectal cancer tissue specimens was performed using an anti-C4.4A-2 polyclonal antibody.
  • Tumor eruption is an indicator of tumor malignancy. Therefore, in order to examine the relationship between the expression pattern of human C4.4A protein in tumor exudation and tumor exudation, tumor exudation was measured. Tumor exudation was evaluated according to the method described in Shinto et al., Dis Colon Rectum 49: 1422-1430 (2006). The outline is described below. A single isolated cancer cell or a cell population consisting of up to 4 cancer cells was defined as tumor enucleation. When observed under a 20 ⁇ objective lens, the cancer tissues were classified into two groups according to the appearance frequency of tumor exudation in the region where tumor exudation was observed at the highest density. A case where the appearance frequency of tumor eruption was 0 to 9 was defined as low-frequency eruption, and a case where the present frequency of tumor eruption was 10 or more was defined as high-frequency eruption.
  • Example 3 Verification of relationship between target peptide and staining pattern ⁇ I. Experimental materials and methods> In the same manner as in Example 1, a polyclonal antibody was obtained from two rabbits different from the anti-C4.4A-2 antibody using the peptide having the amino acid sequence shown in SEQ ID NO: 1 as the target peptide. They were named anti-C4.4A-2-2 polyclonal antibody and anti-C4.4A-2-3 polyclonal antibody, respectively.
  • FIG. 8 shows the site of each target peptide in the human C4.4A protein.
  • the staining pattern by immunohistochemistry in a sample derived from a colon cancer patient was compared with the staining pattern with the anti-C4.4A-2 antibody obtained in Example 1. Immunohistochemistry was performed as in Examples 1 and 2.
  • Example 2 The above results indicate that the staining pattern in the cancer invasion advanced part observed in Example 2 is specifically observed in the antibody targeting a specific site of human C4.4A protein.
  • Example 4 Preparation of monoclonal antibody and verification of specificity ⁇ I. Experimental materials and methods> 1.
  • Preparation of monoclonal antibody A hybridoma producing a monoclonal antibody was obtained according to the following procedure. [1] As a target peptide, a peptide having a sequence with one residue of cysteine added to the N-terminus of the peptide having the amino acid sequence shown in SEQ ID NO: 1 was synthesized. [2] The peptide was conjugated to bovine thyroglobulin and immunized to mice.
  • the immunized animals are: SPF / VAF B6D2F1 / Crlj (BDF1 mouse) male, 2 6-week-old (acquired from Charles River Japan), and SPF / VAF BALB / cAnNCrlCrlj (BALB / c mouse) male, Two, 6 weeks old (obtained from Japan Charles River).
  • the immunization conditions were 50 ⁇ g / animal / dose (initial FCA, second and subsequent FICA emulsions) subcutaneously and intradermally in the abdomen and back once every 7 days for a total of 4 immunizations.
  • Test blood collection was evaluated by antigen solid-phase ELISA and immunohistochemical staining, and individuals with increased antibody titers were selected.
  • Spleen cells and lymph nodes were prepared from the selected mouse individuals and fused with myeloma (P3X63Ag8.653) to obtain fused cells (hybridoma).
  • the obtained fused cells were seeded by limiting dilution. Subsequently, the culture supernatant was subjected to ELISA on an antigen solid phase plate on which the target peptide was immobilized, and hybridomas producing an antibody having a high antigen titer relative to the target titer were selected.
  • One of the obtained hybridomas was named 11A1 and used for the following analysis.
  • the produced hybridoma 11A1 was deposited on April 27, 2011 (original deposit date), which was deposited with the Patent Microorganisms of the National Institute of Technology and Evaluation of the National Institute of Technology and Evaluation, addressed 2-5-8, Kazusa Kamashita, Kisarazu, Chiba, Japan. On May 31, 2011, a request for transfer from the original deposit to a deposit based on the Budapest Treaty was accepted on May 31, 2011.
  • the accession number is NITE BP-1090, and the display for identification given by the trustee is “CA4.4A Hybridoma 11A1”.
  • anti-C4.4A-2 monoclonal antibody Using the monoclonal antibody produced by the hybridoma 11A1 obtained above (hereinafter referred to as anti-C4.4A-2 monoclonal antibody), a staining pattern by immunohistochemistry was performed on a sample derived from a colon cancer patient. The staining pattern was compared with the anti-C4.4A-2 antibody obtained in Example 1.
  • a staining pattern using an anti-C4.4A-2 monoclonal antibody previously adsorbed to an excessive amount of human C4.4A-2 antigenic peptide was also obtained by the following method. [1] Add antibody and peptide to antibody dilution buffer (1% BSA in PBS) so that antibody: peptide is 1 mol: 20 mol. As a control, a solution in which purified water is added instead of the peptide is used. [2] Inverted overnight at 4 ° C.
  • FIG. 11 shows staining patterns of anti-C4.4A-2 monoclonal antibody and anti-C4.4A-2 polyclonal antibody in serial sections of a sample derived from a colon cancer patient.
  • the staining pattern obtained with the anti-C4.4A-2 monoclonal antibody was frequently observed on the cell membrane in the cancer invasion advanced portion.
  • FIG. 12 shows a staining pattern using an anti-C4.4A-2 monoclonal antibody previously adsorbed to an excessive amount of human C4.4A-2 antigenic peptide. No specific staining pattern was observed in advanced cancer invasion.
  • FIG. 13 shows the results of Western blot analysis.
  • a band (a band indicated by an arrow in the figure) could be detected at a position of about 40 kDa as in the case of the anti-C4.4A-2 polyclonal antibody.
  • a band at a position of about 40 kDa is preliminarily adsorbed to an anti-C4.4A-2 polyclonal antibody previously adsorbed to an excess amount of human C4.4A-2 antigen peptide and to an excess amount of human C4.4A-2 antigen peptide.
  • anti-C4.4A-2 monoclonal antibody was used, it was not detected.
  • Example 5 Correlation between human C4.4A protein expression pattern and lymph node metastasis and hematogenous metastasis To verify in more detail the correlation between human C4.4A protein expression pattern and lymph node metastasis and hematogenous metastasis In addition to 122 cases of colorectal cancer tissue specimens used in Example 3, a total of 132 cases including 10 cases (Osaka University Surgery Department) colon cancer tissue specimens with distant metastases were added as in Example 2. Analysis was performed. The results of statistical analysis are shown in Tables 8-13.
  • the prognosis was statistically significantly poor in patients whose cancer tissue donation showed type A expression pattern of human C4.4A protein (Example 2). Further, as a result of detailed analysis, it was suggested that the type A expression pattern of human C4.4A protein correlates with hematogenous metastasis but has little correlation with lymph node metastasis (Example 5). It was also suggested that the type A expression pattern of the human C4.4A protein is a factor that more significantly shows the risk of cancer recurrence than lymph node metastasis.
  • the type A expression pattern of human C4.4A protein is used as a criterion for poor prognosis, and further, the type A expression pattern of human C4.4A protein is a cancer caused by hematogenous metastasis. It is an indicator of recurrence risk.
  • lymphatic metastasis has become an indication of whether or not to perform aftercare to prevent cancer recurrence such as administration of anticancer agents after surgery.
  • lymphatic metastasis is negative, prophylactic aftercare is generally not performed, but cancer is recurrent only in about 10 to 20% of patients with lymphatic metastasis negative.
  • lymphatic metastasis is positive, preventive aftercare such as administration of an anticancer agent is performed, but the rate of cancer recurrence when lymphatic metastasis is positive is about 25 to 40%. This means that when the presence or absence of lymph node metastasis is used as an indicator, patients who do not need preventive aftercare are physically, mentally and economically burdened.
  • the method using the antibody of the present invention can detect the risk of hematogenous metastasis from the type A expression pattern of human C4.4A protein. Therefore, it is considered that cancer recurrence due to hematogenous metastasis, which could not be detected when using the presence or absence of conventional lymph node metastasis as an index, can be predicted.
  • a patient who has a high risk of cancer recurrence after cancer surgery can be predicted with high accuracy. That is, aftercare is not performed in patients who do not require preventive aftercare, and it becomes easy to focus on aftercare in patients who truly need preventive aftercare.

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Abstract

Disclosed are: an antibody which can be used for the simple and reliable determination of the prognosis of a cancer patient; and a method and a kit for determining the prognosis of a cancer patient in a simple manner and with high reliability. Specifically disclosed are: an antibody which can bind specifically to a domain comprising the amino acid sequence represented by SEQ ID NO:1; and a method for determining the prognosis of a cancer patient on the basis of an expression pattern that is detected by an immunoassay using the antibody.

Description

癌予後判定可能な抗体、および癌予後判定方法Antibody capable of determining cancer prognosis, and method for determining cancer prognosis
　本発明は、癌患者の予後を簡便かつ信頼性よく判定するために使用可能な抗体、ならびに癌患者の予後を簡便かつ信頼性よく判定する方法、キットおよび診断薬に主に関する。 The present invention mainly relates to an antibody that can be used for easily and reliably determining the prognosis of a cancer patient, and a method, kit, and diagnostic agent for simply and reliably determining the prognosis of a cancer patient.
　癌は、世界中のヒトの主要な死亡原因の一つである。近年の技術進歩、特に化学療法の分野における進歩にも関わらず、癌による死者数は大変多い。例えば、大腸癌および直腸癌による総死者数は２００９年においては約４９，９２０人に上ると推定されている。 Cancer is one of the leading causes of human death worldwide. Despite recent technological advances, particularly in the field of chemotherapy, the number of deaths from cancer is very high. For example, the total death toll from colorectal and rectal cancer is estimated to be about 49,920 in 2009.
　癌は外科的手術または化学療法などによる治療を行った後も、しばしば再発および転移が起きることが知られている。そのため、治療後も経過を観察しながら、個々の患者に適したアフターケアを行っていくことが臨床的に大変重要である。治療前または治療直後に、癌再発および転移の危険性を簡便かつ信頼性よく判定することができれば、個々の患者の再発・転移の可能性に応じて、可能性が高い患者については慎重な経過観察や重点的な予防的措置を行い、可能性が高くない患者については過度な経過観察や予防的措置を行わないといった、個々の患者に応じたアフターケアを行っていくことが可能となる。 It is known that cancer often recurs and metastasizes even after being treated by surgery or chemotherapy. Therefore, it is clinically important to provide aftercare suitable for individual patients while observing the progress after treatment. If the risk of cancer recurrence and metastasis can be determined easily and reliably before treatment or immediately after treatment, depending on the likelihood of recurrence / metastasis of each individual patient, a cautious course should be followed for patients with high possibility It is possible to perform aftercare according to individual patients, such as observation and prioritized precautionary measures, and patients who are not highly likely not to undergo excessive follow-up and preventive measures.
　このような状況の中で、癌再発および転移の危険性について、すなわち癌患者の予後が良好であるか不良であるかについて、簡便かつ信頼性よく判定する方法が広く求められている。癌再発および転移の危険因子の一つとして、浸潤細胞が知られている。しかし、癌、特に大腸癌において、浸潤細胞、特に血行性転移を生じうる活動性の浸潤細胞のみを検出する方法は、従来知られていなかった。 Under such circumstances, there is a wide demand for a simple and reliable method for determining the risk of cancer recurrence and metastasis, that is, whether the prognosis of a cancer patient is good or bad. Infiltrating cells are known as one of risk factors for cancer recurrence and metastasis. However, a method for detecting only infiltrating cells, particularly active infiltrating cells that can cause hematogenous metastasis in cancer, particularly colorectal cancer, has not been known.
　大腸癌患者、特に手術後の患者において、従来リンパ節転移の有無が、予後の指標として広く用いられてきた。しかしながら、リンパ節転移の有無を指標とした場合、手術後の大腸癌患者が病理所見によりリンパ節転移陰性であっても、癌再発が生じる場合がある。また逆に、リンパ節転移陽性であっても癌再発が生じない場合がある。このように、リンパ節転移の有無を指標とした従来方法の元では、患者に適切な手術後のアフターケアが行えていなかった現状がある。このため、必要がない手術後のアフターケア（例えば、抗癌剤投与など）を患者に行なっている場合が多くあり、患者に対する肉体的、精神的および経済的負担の原因ならびに国などが支出する医療費の増大の原因となっている。 The presence or absence of lymph node metastasis has been widely used as a prognostic indicator in patients with colorectal cancer, especially after surgery. However, when the presence or absence of lymph node metastasis is used as an index, cancer recurrence may occur even if the colon cancer patient after surgery is negative for lymph node metastasis due to pathological findings. Conversely, cancer recurrence may not occur even if the lymph node metastasis is positive. Thus, under the conventional method using the presence or absence of lymph node metastasis as an index, there is a current situation in which aftercare after surgery appropriate for a patient has not been performed. For this reason, there are many cases where unnecessary post-surgery aftercare (for example, administration of anticancer drugs) is given to the patient, causing the physical, mental and economic burden on the patient, and the medical expenses expended by the government, etc. It is a cause of increase.
　一方、グリコシルフォスファチジルイノシトール（ＧＰＩ）アンカー型膜タンパク質であるＣ４．４Ａタンパク質は、当初、ラット由来の高度転移性の膵臓癌細胞において発現し、通常の癌細胞には発現しないタンパク質として見出された。ラットＣ４．４Ａタンパク質と相同なヒトＣ４．４Ａタンパク質が存在し同定されている（非特許文献１参照）。しかし、ヒトＣ４．４Ａタンパク質の生理学的機能の多くは明らかにされていない。 On the other hand, C4.4A protein, which is a glycosylphosphatidylinositol (GPI) -anchored membrane protein, was initially expressed in rat-derived highly metastatic pancreatic cancer cells and was found not to be expressed in normal cancer cells. It was done. A human C4.4A protein homologous to the rat C4.4A protein exists and has been identified (see Non-Patent Document 1). However, many of the physiological functions of human C4.4A protein have not been clarified.
　近年の研究結果より、ヒト悪性腫瘍の一部においても、ヒトＣ４．４Ａ遺伝子の発現が見出されている（非特許文献２～４参照）。さらに、ヒトＣ４．４Ａ遺伝子転写産物の発現が、非小細胞肺癌患者の予後不良と相関しているという臨床結果もある（非特許文献５参照）。しかし、ヒトＣ４．４Ａタンパク質を、どのようにして癌の予後を簡便かつ信頼性よく判定することに用い得るかは、全く知られていない。 From recent research results, the expression of the human C4.4A gene has been found in some human malignant tumors (see Non-Patent Documents 2 to 4). Furthermore, there is a clinical result that the expression of the human C4.4A gene transcript is correlated with poor prognosis in patients with non-small cell lung cancer (see Non-Patent Document 5). However, it is not known at all how the human C4.4A protein can be used to easily and reliably determine the prognosis of cancer.
　上記のように、従来技術では、癌の浸潤細胞を特異的に検出することができないのが現状である。そこで、本発明は、上記従来技術の課題を解決することを目的とする。具体的には、癌の浸潤細胞を特異的に検出することで癌患者の予後を簡便かつ信頼性よく判定するために使用可能な抗体、ならびに癌患者の予後を簡便かつ信頼性よく判定する方法、キット、および診断薬を提供することを主な目的とする。 As described above, in the conventional technology, cancer infiltrating cells cannot be specifically detected. Accordingly, an object of the present invention is to solve the above-described problems of the conventional art. Specifically, antibodies that can be used to easily and reliably determine the prognosis of cancer patients by specifically detecting cancer infiltrating cells, and methods for easily and reliably determining the prognosis of cancer patients The main objective is to provide kits and diagnostics.
　本発明者らは、上記課題を解決すべく鋭意検討したところ、ヒトＣ４．４Ａタンパク質のアミノ末端（Ｎ末端）部位を認識する抗体は癌細胞においてヒトＣ４．４Ａタンパク質の発現を検出することができないが、ヒトＣ４．４Ａタンパク質のカルボキシル末端（Ｃ末端）部位の特定配列を認識する抗体は癌細胞においてヒトＣ４．４Ａタンパク質の発現を検出可能であることを見出した。さらに、前記ヒトＣ４．４Ａタンパク質のＣ末端部位の特定配列を認識する抗体を用いて、ヒトＣ４．４Ａタンパク質が癌の浸潤細胞に特異的に発現する様子を検出することが可能であり、かつヒトＣ４．４Ａタンパク質の発現パターンが癌患者の予後と高く相関することを見出した。本発明は、かかる知見に基づいてさらに改良を重ねることによって完成したものである。 As a result of intensive studies to solve the above problems, the present inventors have found that an antibody that recognizes the amino terminal (N-terminal) site of human C4.4A protein can detect the expression of human C4.4A protein in cancer cells. However, it was found that an antibody that recognizes a specific sequence at the carboxyl terminal (C-terminal) site of human C4.4A protein can detect the expression of human C4.4A protein in cancer cells. Furthermore, using an antibody that recognizes a specific sequence at the C-terminal site of the human C4.4A protein, it is possible to detect the state in which the human C4.4A protein is specifically expressed in cancer infiltrating cells, and It was found that the expression pattern of human C4.4A protein is highly correlated with the prognosis of cancer patients. The present invention has been completed by making further improvements based on such findings.
　すなわち、本発明は。下記態様の発明を包含する：
項１．配列番号１に示すアミノ酸配列の領域に特異的に結合する抗ヒトＣ４．４Ａタンパク質抗体。
項２．ポリクローナル抗体である、項１に記載の抗体。
項３．モノクローナル抗体である、項１に記載の抗体
項４．受託番号：NITE BP-1090で特定されるハイブリドーマが産生するモノクローナル抗体である、項３に記載のモノクローナル抗体。
項５－１．癌患者の予後を判定する方法であって、
（１）癌患者から採取した癌組織において、項１から４のいずれかに記載の抗体でヒトＣ４．４Ａタンパク質の発現を検出する工程、
（２）前記工程（１）の検出結果に基づいて、癌浸潤先進部の細胞において、細胞膜と細胞質との間でヒトＣ４．４Ａタンパク質の局在を比較し、かつ癌浸潤先進部と癌病巣の表層部または中層部との間で、ヒトＣ４．４Ａタンパク質の発現量を比較する工程、および
（３）前記工程（２）の比較に基づいて、前記ヒトＣ４．４Ａタンパク質が前記癌浸潤先進部の細胞の細胞膜上に集積し、かつ前記ヒトＣ４．４Ａタンパク質の発現量が前記癌浸潤先進部の細胞において、前記癌病巣の表層部または中層部と比べて亢進している場合は、予後不良と判定する工程、を含む方法。
項５－２．癌患者の予後を判定する方法であって、
（１）癌患者から採取した癌組織において、項１から４のいずれかに記載の抗体を用いて免疫染色を行なう工程、
（２）前記工程（１）の免疫染色に基づいて、癌浸潤先進部の細胞において、細胞膜と細胞質との間でヒトＣ４．４Ａタンパク質の染色強度を比較し、かつ癌浸潤先進部と癌病巣の表層部または中層部との間で、ヒトＣ４．４Ａタンパク質の染色強度を比較する工程、および
（３）前記工程（２）の染色強度の比較に基づいて、前記ヒトＣ４．４Ａタンパク質の染色強度が、前記癌浸潤先進部の細胞の細胞膜において、前記癌浸潤先進部の細胞の細胞質と比べて亢進、かつ前記ヒトＣ４．４Ａタンパク質の染色強度が前記癌浸潤先進部の細胞において、前記癌病巣の表層部または中層部と比べて亢進している場合は、予後不良と判定する工程、を含む方法。
項５－３．癌患者の予後を判定する方法であって、
（１）癌患者から採取した癌組織を、前記抗体でヒトＣ４．４Ａタンパク質について免疫測定を行う工程、
（２）前記工程（１）の免疫測定の結果について、癌浸潤先進部と癌病巣の表層部または中層部との間で、測定結果を比較する工程、および
（３）前記工程（２）の測定結果の比較に基づいて、前記ヒトＣ４．４Ａタンパク質が前記癌浸潤先進部の細胞の細胞膜上に集積し、かつ前記ヒトＣ４．４Ａタンパク質の発現量が前記癌浸潤先進部の細胞において前記癌病巣の表層部または中層部の細胞と比べて亢進している場合は、予後不良と判定する工程、を含む方法。
項５－４．癌患者の癌再発リスクを判定する方法であって、
（１）癌患者から採取した癌組織において、項１から４のいずれかに記載の抗体でヒトＣ４．４Ａタンパク質の発現を検出する工程、
（２）前記工程（１）の検出結果に基づいて、癌浸潤先進部の細胞において、細胞膜と細胞質との間でヒトＣ４．４Ａタンパク質の局在を比較し、かつ癌浸潤先進部と癌病巣の表層部または中層部との間で、ヒトＣ４．４Ａタンパク質の発現量を比較する工程、および
（３）前記工程（２）の比較に基づいて、前記ヒトＣ４．４Ａタンパク質が前記癌浸潤先進部の細胞の細胞膜上に集積し、かつ前記ヒトＣ４．４Ａタンパク質の発現量が前記癌浸潤先進部の細胞において、前記癌病巣の表層部または中層部と比べて亢進している場合は、予後不良と判定する工程、を含む方法。
項６．予後が、癌再発リスクに基づく予後である、項５－１、項５－２または項５－３のいずれか１項に記載の方法。
項７．癌再発が、血行性転移に起因する癌再発である、項５－４または項６に記載の方法。
項８－１．癌患者が消化器系の癌の患者である、項５－１項、項５－２、項５－３、項５－４、項６または項７のいずれか１項に記載の方法。
項８－２．癌患者が大腸癌患者である、項５－１項、項５－２、項５－３、項５－４、項６または項７のいずれか１項に記載の方法。
項９―１．癌患者の予後を判定するためのキットであって、項１から４のいずれかに記載の抗体を含んでなるキット。
項９―２．癌患者の癌再発リスクを判定するためのキットであって、項１から４のいずれかに記載の抗体を含んでなるキット。
項１０．予後が、癌再発リスクに基づく予後である、項９―１に記載のキット。
項１１．癌再発が、血行性転移に起因する癌再発である、項９―２または項１０に記載のキット。
項１２－１．癌患者が消化器系の癌の患者である、項９－１、項９－２、項１０または項１１のいずれか１項に記載のキット。
項１２－２．癌患者が大腸癌患者である、項９－１、項９－２、項１０または項１１のいずれか１項に記載のキット。
項１３－１．項１から４のいずれかに記載の抗体を含む、癌再発リスクの診断試薬。
項１３－２．項１から４のいずれかに記載の抗体を含む、癌診断試薬。
項１４．癌再発が、血行性転移に起因する癌再発である、項１３－１または項１３－２に記載の診断試薬。
項１５－１．癌が消化器系の癌の患である、項１３－１、項１３－２または項１４のいずれか１項に記載の診断試薬
項１５－２．癌が大腸癌である、項１３－１、項１３－２または項１４のいずれか１項に記載の診断試薬。 That is, the present invention. Including the following aspects of the invention:
Item 1. An anti-human C4.4A protein antibody that specifically binds to a region of the amino acid sequence shown in SEQ ID NO: 1.
Item 2. Item 2. The antibody according to Item 1, which is a polyclonal antibody.
Item 3. Item 3. The antibody according to Item 1, which is a monoclonal antibody. Item 6. The monoclonal antibody according to Item 3, which is a monoclonal antibody produced by a hybridoma specified by NITE BP-1090.
Item 5-1. A method for determining the prognosis of a cancer patient,
(1) a step of detecting expression of a human C4.4A protein with the antibody according to any one of Items 1 to 4 in a cancer tissue collected from a cancer patient;
(2) Based on the detection result of the step (1), the localization of the human C4.4A protein is compared between the cell membrane and the cytoplasm in the cells of the advanced cancer invasion, and the advanced cancer invasion and the cancer lesion are compared. Comparing the expression level of the human C4.4A protein with the surface layer portion or the middle layer portion of (3), and (3) based on the comparison in the step (2), the human C4.4A protein is Prognosis when the expression level of the human C4.4A protein is accumulated on the cell membrane of some cells and is increased in the cells in the cancer invasion advanced portion as compared with the surface layer portion or middle layer portion of the cancer lesion. Determining a failure.
Item 5-2. A method for determining the prognosis of a cancer patient,
(1) performing immunostaining using the antibody according to any one of Items 1 to 4 in a cancer tissue collected from a cancer patient;
(2) Based on the immunostaining in the above step (1), the staining intensity of human C4.4A protein is compared between the cell membrane and the cytoplasm in the cells of the advanced cancer invasion, and the advanced cancer invasion and the cancer lesion are compared. Comparing the staining intensity of the human C4.4A protein with the surface layer portion or the middle layer portion of the above, and (3) staining of the human C4.4A protein based on the comparison of the staining intensity of the step (2). Intensity is enhanced in the cell membrane of the advanced cancer invasion cell compared to the cytoplasm of the advanced cancer invasion cell, and the staining intensity of the human C4.4A protein is increased in the advanced cancer invasion cell. And a step of determining that the prognosis is poor when the lesion is enhanced compared to the surface layer or middle layer of the lesion.
Item 5-3. A method for determining the prognosis of a cancer patient,
(1) A step of immunoassaying a cancer tissue collected from a cancer patient with respect to the human C4.4A protein with the antibody,
(2) About the result of the immunoassay in the step (1), a step of comparing the measurement result between the advanced portion of cancer invasion and the surface layer portion or middle layer portion of the cancer lesion, and (3) of the step (2) Based on the comparison of the measurement results, the human C4.4A protein is accumulated on the cell membrane of the cancer invasion advanced portion cell, and the expression level of the human C4.4A protein is increased in the cancer invasion advanced portion cell. And a step of determining that the prognosis is poor when cells are elevated compared to cells in the surface layer or middle layer of the lesion.
Item 5-4. A method for determining the risk of cancer recurrence in a cancer patient,
(1) a step of detecting expression of a human C4.4A protein with the antibody according to any one of Items 1 to 4 in a cancer tissue collected from a cancer patient;
(2) Based on the detection result of the step (1), the localization of the human C4.4A protein is compared between the cell membrane and the cytoplasm in the cells of the advanced cancer invasion, and the advanced cancer invasion and the cancer lesion are compared. Comparing the expression level of the human C4.4A protein with the surface layer portion or the middle layer portion of (3), and (3) based on the comparison in the step (2), the human C4.4A protein is Prognosis when the expression level of the human C4.4A protein is accumulated on the cell membrane of some cells and is increased in the cells in the cancer invasion advanced portion as compared with the surface layer portion or middle layer portion of the cancer lesion. Determining a failure.
Item 6. The method according to any one of Items 5-1, 5-2, or 5-3, wherein the prognosis is a prognosis based on a risk of cancer recurrence.
Item 7. Item 7. The method according to Item 5-4 or Item 6, wherein the cancer recurrence is cancer recurrence due to hematogenous metastasis.
Item 8-1. Item 8. The method according to any one of Items 5-1, 5-2, 5-3, 5-4, 6 or 7, wherein the cancer patient is a patient with cancer of the digestive system.
Item 8-2. Item 8. The method according to any one of Items 5-1, 5-2, 5-3, 5-4, 6 or 7, wherein the cancer patient is a colorectal cancer patient.
Item 9-1. A kit for determining the prognosis of a cancer patient, comprising the antibody according to any one of Items 1 to 4.
Item 9-2. A kit for determining the risk of cancer recurrence in a cancer patient, comprising the antibody according to any one of Items 1 to 4.
Item 10. Item 9. The kit according to Item 9-1, wherein the prognosis is a prognosis based on the risk of cancer recurrence.
Item 11. Item 10. The kit according to Item 9.2 or Item 10, wherein the cancer recurrence is cancer recurrence due to hematogenous metastasis.
Item 12-1. Item 12. The kit according to any one of Items 9-1, 9-2, 10 or 11, wherein the cancer patient is a patient with cancer of the digestive system.
Item 12-2. Item 12. The kit according to any one of Items 9-1, 9-2, 10 or 11, wherein the cancer patient is a colorectal cancer patient.
Item 13-1. A diagnostic reagent for cancer recurrence risk, comprising the antibody according to any one of Items 1 to 4.
Item 13-2. Item 5. A cancer diagnostic reagent comprising the antibody according to any one of Items 1 to 4.
Item 14. Item 13. The diagnostic reagent according to Item 13-1 or Item 13-2, wherein the cancer recurrence is cancer recurrence due to hematogenous metastasis.
Item 15-1. Item 15. The diagnostic reagent according to any one of Items 13-1, 13-2, or 14, wherein the cancer is a disease of a cancer of the digestive system. Item 15. The diagnostic reagent according to any one of Items 13-1, 13-2, or 14, wherein the cancer is colorectal cancer.
　本発明の抗体は、癌浸潤細胞において特異的に発現するヒトＣ４．４Ａタンパク質を検出することができる。 The antibody of the present invention can detect human C4.4A protein specifically expressed in cancer infiltrating cells.
　また、本発明の抗体を用いて癌検体におけるヒトＣ４．４Ａタンパク質の発現パターンを測定することで、癌患者の予後判定を簡便かつ信頼性よく行うことができる。かかる予後判定に基づいて、個々の癌患者に適した個々の患者に適したアフターケアを行っていくことができる。 Further, by measuring the expression pattern of human C4.4A protein in a cancer specimen using the antibody of the present invention, it is possible to easily and reliably determine the prognosis of cancer patients. Based on such prognosis determination, aftercare suitable for individual patients suitable for individual cancer patients can be performed.
　さらに、本発明のキットまたは癌診断試薬を用いて、前記の判定方法を容易に行うことが可能となる。 Furthermore, the determination method can be easily performed using the kit or the cancer diagnostic reagent of the present invention.
　本発明を用いることで、特に大腸がんなどの患者において、抗癌剤投与などの必要なアフターケアの要否について、精度が高い判定が可能となる。その結果、患者への負担となる治療を抑制し、ひいては社会保障費の削減へとつながることが期待される。 By using the present invention, it is possible to determine with high accuracy whether or not necessary aftercare such as administration of an anticancer drug is necessary particularly in patients with colorectal cancer and the like. As a result, it is expected that the treatment that burdens the patient will be suppressed, leading to a reduction in social security costs.
図１は、ヒトＣ４．４Ａタンパク質のアミノ酸配列、および抗原として用いた２つの配列（太字）を示す。ＧＰＩアンカー付加シグナル配列は、四角で囲って示した。FIG. 1 shows the amino acid sequence of the human C4.4A protein and the two sequences (bold) used as antigens. The GPI anchor addition signal sequence is shown enclosed in a box.
図２は、正常な食道組織検体、大腸がん由来細胞株（ＨＣＴ１１６、ＫＭ１２ＳＭ）試料、および３例の患者から採取した正常検体（Ｎ）および大腸がん組織検体（Ｔ）について、ヒトＣ４．４Ａタンパク質のウェスタンブロット解析の結果を示す。食道上皮組織検体では、抗Ｃ４．４Ａ－１ポリクローナル抗体および抗Ｃ４．４Ａ－２ポリクローナル抗体は、分子量６７ｋＤａ近傍に明確なバンドを示した。しかし、大腸がん由来細胞株試料と大腸組織検体の両方について、抗Ｃ４．４Ａ－２ポリクローナル抗体のみが約４０ｋＤａの位置に顕著なバンドを示し、一方で抗Ｃ４．４Ａ－１ポリクローナル抗体はバンドを示さなかった。アクチンの検出は、ローディングコントロールである。FIG. 2 shows human C4.. For normal esophageal tissue specimens, colon cancer-derived cell lines (HCT116, KM12SM) samples, and normal specimens (N) and colon cancer tissue specimens (T) collected from three patients. The result of the Western blot analysis of 4A protein is shown. In the esophageal epithelial tissue specimen, the anti-C4.4A-1 polyclonal antibody and the anti-C4.4A-2 polyclonal antibody showed a clear band around a molecular weight of 67 kDa. However, for both colorectal cancer-derived cell line samples and colorectal tissue samples, only the anti-C4.4A-2 polyclonal antibody shows a prominent band at a position of about 40 kDa, while the anti-C4.4A-1 polyclonal antibody Did not show. Detection of actin is a loading control.
図３は、正常な食道粘膜の組織切片（Ａ、Ｂ）および大腸癌組織切片（Ｃ、Ｄ）について、抗Ｃ４．４Ａ－１ポリクローナル抗体（Ａ，Ｃ）および抗Ｃ４．４Ａ－２ポリクローナル抗体（Ｂ、Ｄ）を用いた免疫組織化学の結果を示す。（Ａ、Ｂ）基底上の扁平上皮において染色が観察された。（Ｃ）大腸癌組織検体においては、抗Ｃ４．４Ａ－１ポリクローナル抗体は何らシグナルが検出されなかった。（Ｄ）同じ試料において抗Ｃ４．４Ａ－２抗体では染色シグナルが得られた。倍率：Ａ、Ｂ：×１００；Ｃ、Ｄ：×４０。FIG. 3 shows anti-C4.4A-1 polyclonal antibody (A, C) and anti-C4.4A-2 polyclonal antibody for normal esophageal mucosa tissue sections (A, B) and colon cancer tissue sections (C, D). The result of immunohistochemistry using (B, D) is shown. (A, B) Staining was observed in the squamous epithelium on the base. (C) No signal was detected in the anti-C4.4A-1 polyclonal antibody in the colon cancer tissue specimen. (D) A staining signal was obtained with the anti-C4.4A-2 antibody in the same sample. Magnification: A, B: x100; C, D: x40.
図４は、抗Ｃ４．４Ａ－２ポリクローナル抗体を用いた、ヒトＣ４．４Ａタンパク質の発現についての免疫組織化学解析の結果を示す。（Ａ）大腸癌組織検体の癌浸潤先進部および浸潤部位における、ヒトＣ４．４Ａタンパク質の特異的な発現パターンを示す。癌病巣の表層部または中層部においては、ヒトＣ４．４Ａタンパク質の弱い発現がほとんどの場合は細胞質において検出され、一方癌浸潤先進部においては高頻度で細胞膜上にＣ４．４Ａタンパク質の発現が検出された。膨大な数の癌組織検体のうちで、新生の間質に向かって面した細胞のみがヒトＣ４．４Ａタンパク質を発現していた（左下パネル）。倍率：左上：×１２．５；左下×４０；右上、右下：×１００。（Ｂ）腫瘍蔟出における免疫染色の結果を示す。蔟出細胞は、大腸癌組織の癌浸潤先進部から脱落している。これはＡ型の発現パターンとして分類した。倍率：×１００。（Ｃ）蔟出頻度と、癌浸潤先進部におけるヒトＣ４．４Ａタンパク質の発現パターンとの間に有意な相関関係が認められた（Ｐ＜０．０００１）。FIG. 4 shows the results of immunohistochemical analysis of human C4.4A protein expression using anti-C4.4A-2 polyclonal antibody. (A) A specific expression pattern of human C4.4A protein in a cancer invasion advanced part and an invasion site of a colon cancer tissue specimen is shown. In the surface layer or middle layer of the cancer lesion, weak expression of human C4.4A protein is detected in the cytoplasm in most cases, while in the cancer invasion advanced portion, the expression of C4.4A protein is frequently detected on the cell membrane. It was done. Of the enormous number of cancer tissue specimens, only the cells facing the neoplastic stroma expressed human C4.4A protein (lower left panel). Magnification: upper left: x12.5; lower left x40; upper right, lower right: x100. (B) shows the result of immunostaining in tumor exudation. The exudate cells have fallen from the cancer invasion advanced part of the colon cancer tissue. This was classified as a type A expression pattern. Magnification: x100. (C) A significant correlation was found between the frequency of sputum and the expression pattern of human C4.4A protein in advanced cancer invasion (P <0.0001).
図５は、リンパ節転移部位および肺浸潤部位における、抗Ｃ４．４Ａ－２ポリクローナル抗体による免疫組織化学の結果を示す。（Ａ）リンパ節転移部位：繊維性被膜に浸潤する浸潤性腫瘍細胞が特異的に、ヒトＣ４．４Ａタンパク質を強く、細胞膜上に発現していた。（Ｂ）肺浸潤部位：浸潤周縁部（矢印部位）における浸潤性腫瘍細胞は、同様な発現パターンを示した。倍率：左上：×１２．５；左下：×４０；右上、右下：×１００。FIG. 5 shows the results of immunohistochemistry with anti-C4.4A-2 polyclonal antibody at the lymph node metastasis site and the lung infiltration site. (A) Lymph node metastasis site: Infiltrating tumor cells that infiltrate the fibrous cap were specifically expressed with strong human C4.4A protein on the cell membrane. (B) Lung infiltration site: Infiltrating tumor cells in the periphery of the infiltration (arrow site) showed a similar expression pattern. Magnification: upper left: × 12.5; lower left: × 40; upper right, lower right: × 100.
図６は、ヒトＣ４．４Ａタンパク質の発現パターンと、患者の予後との関係（５年間全生存率）について示す。癌のステージがＩ期～ＩＶ期の癌患者群（ｎ＝１２２）についての５年間全生存率を表すカプランマイヤープロットを示す。ヒトＣ４．４Ａタンパク質の局在の変化（Ａ＋Ｂ１）または発現強度の変化（Ａ＋Ｂ２）のいずれとも、より短い５年間全生存率（ＯＳ）と相関する（Ｐ＝０．０１４およびＰ＝０．００６）。より顕著な差が、Ａ型と他の型の全体（Ｂ１＋Ｂ２＋Ｃ）との間で認められた（Ｐ＝０．０００９）。一方で、Ｂ１型およびＢ２型は、Ｃ型との間に顕著な差は認められなかった。FIG. 6 shows the relationship between the expression pattern of human C4.4A protein and the prognosis of patients (5-year overall survival rate). FIG. 2 shows a Kaplan-Meier plot representing the 5-year overall survival rate for a group of cancer patients whose stage of cancer is stage I to stage IV (n = 122). Either a change in the localization of the human C4.4A protein (A + B1) or a change in expression intensity (A + B2) correlates with a shorter 5-year overall survival (OS) (P = 0.014 and P = 0.006) ). A more prominent difference was observed between type A and all other types (B1 + B2 + C) (P = 0.0009). On the other hand, the B1 type and the B2 type were not significantly different from the C type.
図７は、ヒトＣ４．４Ａタンパク質の発現パターンと、患者の予後との関係（５年間無病生存率）について示す。癌のステージがＩＩ期およびＩＩＩ期の患者群（ｎ＝８２）についての５年間無病生存率を示す。ステージＩＩ期およびＩＩＩ期の患者群について外科治療後の５年間無病生存率について解析したところ、５年間全生存率と同様の結果が得られた（それぞれについて、Ｐ＝０．０１１、Ｐ＝０．００１６、およびＰ＝０．０００４）。FIG. 7 shows the relationship between the expression pattern of human C4.4A protein and the prognosis of patients (5-year disease-free survival rate). Figure 5 shows 5-year disease free survival for a group of patients with stage II and stage III cancer (n = 82). Analysis of stage II and stage III patient group survival rates after surgery for 5 years yielded results similar to the overall survival rates for 5 years (P = 0.011, P = 0 for each). .0016, and P = 0.004).
図８は、ヒトＣ４．４Ａタンパク質のアミノ酸配列、および抗原として用いた２つの配列（太字）を示す。ＧＰＩアンカー付加シグナル配列は、四角で囲って示した。(i)：Ｃ４．４Ａ－１（配列番号２）；(ii)：Ｃ４．４Ａ－２（配列番号１）；(iii)：Ｃ４．４Ａ（１１９）（配列番号４）；(iv)：Ｃ４．４Ａ（２７７）（配列番号５）。FIG. 8 shows the amino acid sequence of human C4.4A protein and the two sequences (bold) used as antigens. The GPI anchor addition signal sequence is shown enclosed in a box. (i): C4.4A-1 (SEQ ID NO: 2); (ii): C4.4A-2 (SEQ ID NO: 1); (iii): C4.4A (119) (SEQ ID NO: 4); (iv): C4.4A (277) (SEQ ID NO: 5).
図９は、大腸癌組織検体（浸潤先進部）の連続切片における、抗Ｃ４．４Ａ－２ポリクローナル抗体、抗Ｃ４．４Ａ－２―２ポリクローナル抗体および抗Ｃ４．４Ａ－２―３ポリクローナル抗体を用いた免疫組織化学解析の結果を示す。倍率：×１０。FIG. 9 shows the use of anti-C4.4A-2 polyclonal antibody, anti-C4.4A-2-2 polyclonal antibody and anti-C4.4A-2-3 polyclonal antibody in serial sections of colorectal cancer tissue specimen (advanced advanced part). The results of immunohistochemical analysis were shown. Magnification: x10.
図１０は、大腸癌組織検体の浸潤先進部（Invasive front）（右列および中央列）ならびに中層部（Intermediate portion）（左列）の連続切片における、抗Ｃ４．４Ａ－２ポリクローナル抗体（図中、Ｃ４．４Ａ－２上段。）、抗Ｃ４．４Ａ（１１９）ポリクローナル抗体（図中、Ｃ４．４Ａ（１１９）、中段。）および抗Ｃ４．４Ａ（２７７）ポリクローナル抗体（図中、Ｃ４．４Ａ（１１９）、下段。）を用いた免疫組織化学解析の結果を示す。倍率：×１０。FIG. 10 shows anti-C4.4A-2 polyclonal antibody (in the figure) in serial sections of the invasion advanced part (Invasive front) (right column and middle column) and middle layer part (Intermediate portion) (left column) of colorectal cancer tissue specimens. , C4.4A-2, upper panel), anti-C4.4A (119) polyclonal antibody (in the figure, C4.4A (119), middle panel) and anti-C4.4A (277) polyclonal antibody (in the figure, C4.4A). (119), lower panel) shows the results of immunohistochemical analysis. Magnification: x10.
図１１ＡおよびＢは、大腸癌組織検体（浸潤先進部）における、抗Ｃ４．４Ａ－２ポリクローナル抗体（左側、polyclonal）および抗Ｃ４．４Ａ－２モノクローナル抗体（右側、monoclonal）を用いた免疫組織化学解析の結果を示す。抗Ｃ４．４Ａ－２モノクローナル抗体を用いた場合に、抗Ｃ４．４Ａ－２ポリクローナル抗体と同様に、癌浸潤先進部においては細胞膜上にＣ４．４Ａタンパク質の発現が検出された。倍率：×２０。FIGS. 11A and B show immunohistochemistry using anti-C4.4A-2 polyclonal antibody (left, polyclonal) and anti-C4.4A-2 monoclonal antibody (right, monoclonal) in a colon cancer tissue specimen (advanced advanced part). The result of the analysis is shown. When the anti-C4.4A-2 monoclonal antibody was used, the expression of C4.4A protein was detected on the cell membrane at the advanced cancer invasion portion, as with the anti-C4.4A-2 polyclonal antibody. Magnification: x20.
図１２ＡおよびＢは、大腸癌組織検体の連続切片における、抗Ｃ４．４Ａ－２モノクローナル抗体（左側、mAb）および過剰量の標的ペプチドで予め処理した抗Ｃ４．４Ａ－２モノクローナル抗体（右側、mAb + peptide）を用いた免疫組織化学解析の結果を示す。ＡおよびＢは、それぞれ異なる連続切片試料を用いた結果を示す。過剰量の標的ペプチドで予め処理した抗Ｃ４．４Ａ－２モノクローナル抗体を用いた場合には、染色が得られなかった。倍率：×２０。FIGS. 12A and B show anti-C4.4A-2 monoclonal antibody (left side, mAb) and anti-C4.4A-2 monoclonal antibody (right side, mAb) pre-treated with excess target peptide in serial sections of colon cancer tissue specimens. The results of immunohistochemical analysis using (+ peptide) are shown. A and B show the results using different serial slice samples. No staining was obtained when anti-C4.4A-2 monoclonal antibody pretreated with an excess amount of target peptide was used. Magnification: x20.
図１３は、ウェスタンブロット解析の結果を示す。一次抗体として、抗Ｃ４．４Ａ－２ポリクローナル抗体（Polyclonal、Ab）および抗Ｃ４．４Ａ－２モノクローナル抗体（Monoclonal、Ab）、ならびに、過剰量のヒトＣ４．４Ａ－２抗原ペプチドにあらかじめ吸着させたこれらの抗体（Ab + peptide）を用いた。抗Ｃ４．４Ａ－２モノクローナル抗体を用いて、抗Ｃ４．４Ａ－２ポリクローナル抗体と同様に約４０ｋＤａの位置にバンド（図中、矢印で指し示すバンド）を検出することができた。アクチンの検出は、ローディングコントロールである。*で指し示すバンドは、非特異的に検出されるバンドである。FIG. 13 shows the results of Western blot analysis. Anti-C4.4A-2 polyclonal antibody (Polyclonal, Ab) and anti-C4.4A-2 monoclonal antibody (Monoclonal, Ab) as a primary antibody, and pre-adsorbed to an excess amount of human C4.4A-2 antigenic peptide These antibodies (Ab + peptide) were used. Using the anti-C4.4A-2 monoclonal antibody, a band (a band indicated by an arrow in the figure) could be detected at a position of about 40 kDa as in the case of the anti-C4.4A-2 polyclonal antibody. Detection of actin is a loading control. The band indicated by * is a band detected nonspecifically.
　本発明は、癌患者の予後を簡便かつ信頼性よく判定するための抗体、方法、およびキットを提供することを主な目的とする。以下、抗体、方法、キット、診断薬の順に説明する。 The main object of the present invention is to provide antibodies, methods, and kits for easily and reliably determining the prognosis of cancer patients. Hereinafter, the antibody, method, kit, and diagnostic agent will be described in this order.
　１．抗体
　本発明の抗体は、配列番号１に示すアミノ酸配列の領域に特異的に結合する抗体である。　 1. Antibody The antibody of the present invention is an antibody that specifically binds to the region of the amino acid sequence shown in SEQ ID NO: 1.
　配列番号１に示すアミノ酸配列は、配列番号３に示すヒトＣ４．４Ａタンパク質全長配列のうち、３０１～３１６番目の領域である。ここで、ヒトＣ４．４Ａタンパク質とは、配列番号３に示すアミノ酸配列を有するタンパク質、または配列番号１に示すアミノ酸配列の少なくとも１～８番目のアミノ酸配列を含む、配列番号３の一部分のアミノ酸配列を有するタンパク質のいずれをも指すものとする。 The amino acid sequence shown in SEQ ID NO: 1 is the 301st to 316th region of the full-length human C4.4A protein shown in SEQ ID NO: 3. Here, the human C4.4A protein is a protein having the amino acid sequence shown in SEQ ID NO: 3, or an amino acid sequence of a part of SEQ ID NO: 3 containing at least the first to eighth amino acid sequences of the amino acid sequence shown in SEQ ID NO: 1. Any protein that has
　本抗体が配列番号１に示すアミノ酸配列の領域に特異的に結合することは、公知の方法で可視化することができる。例えば、試験管内の試料を対象とするイムノアッセイ法（例えば、ＥＬＩＳＡ法）、免疫電気泳動法（例えばウェスタンブロット法）、および組織または細胞を対象とする免疫組織化学などが例として挙げられるが、これらに限定されるものではない。可視化にあたっては、本発明の抗体が蛍光物質（例えば、フルオレセインイソチアネート）、放射性同位体（例えば、ヨウ素１２５）、酵素（例えば、アルカリフォスファターゼ、西洋ワサビペルオキシターゼ）、その他のタンパク質（例えば、アビジン）などの分子によって標識化されていてもよい。あるいは、本発明の抗体を特異的に認識する二次抗体を用いてもよい。 The specific binding of this antibody to the region of the amino acid sequence shown in SEQ ID NO: 1 can be visualized by a known method. Examples include immunoassay methods (for example, ELISA method) for samples in vitro, immunoelectrophoresis methods (for example, Western blot method), and immunohistochemistry for tissues or cells. It is not limited to. For visualization, the antibody of the present invention is a fluorescent substance (for example, fluorescein isothiocyanate), a radioisotope (for example, iodine 125), an enzyme (for example, alkaline phosphatase, horseradish peroxidase), and other proteins (for example, avidin). It may be labeled with a molecule such as Alternatively, a secondary antibody that specifically recognizes the antibody of the present invention may be used.
　本発明の抗体は、上記配列番号１に示すアミノ酸配列の領域と特異的に結合できる限りその免疫グロブリンクラスについては限定されるものではなく、例えばＩｇＧ、ＩｇＭ、ＩｇＡ、ＩｇＤ、ＩｇＥ、ＩｇＹなどが挙げられる。さらに、そのサブクラスも制限されない。好ましくは、ＩｇＧである。 The antibody of the present invention is not limited in its immunoglobulin class as long as it can specifically bind to the amino acid sequence region shown in SEQ ID NO: 1. For example, IgG, IgM, IgA, IgD, IgE, IgY and the like can be used. Can be mentioned. Furthermore, the subclass is not limited. Preferably, it is IgG.
　本発明の抗体は、抗体を産生しうる哺乳類（例えば、ヒト、マウス、ラット、ウサギ）または鳥類（例えば、ニワトリ、ダチョウ）由来である。 The antibody of the present invention is derived from a mammal (for example, human, mouse, rat, rabbit) or bird (for example, chicken or ostrich) capable of producing the antibody.
　本発明の抗体は、ポリクローナル抗体であってもよいし、モノクローナル抗体であってもよい。さらには、モノクローナル抗体のうち、キメラ抗体、ヒト化抗体、またはヒト抗体であってもよい。ここで、キメラ抗体およびヒト化抗体とは、免疫グロブリンタンパク質のうち、抗原と結合可能な部位以外がヒト由来である抗体を指す。ヒト抗体は、ヒト由来の抗体、または完全なヒト由来の免疫グロブリン遺伝子を有するヒト抗体産生マウス由来である抗体を指す。キメラ抗体、ヒト化抗体、およびヒト抗体は、ヒトにおける免疫原性が低く、抗体を医療製剤などに用いるに当たって有用である。従って、本発明の抗体を医療製剤などに用いる場合にあっては、モノクローナル抗体が好ましく、ヒト化抗体またはヒト抗体が特に好ましい。 The antibody of the present invention may be a polyclonal antibody or a monoclonal antibody. Furthermore, among monoclonal antibodies, a chimeric antibody, a humanized antibody, or a human antibody may be used. Here, the chimeric antibody and the humanized antibody refer to an antibody derived from a human other than a site capable of binding to an antigen among immunoglobulin proteins. A human antibody refers to a human-derived antibody or an antibody derived from a human antibody-producing mouse having a complete human-derived immunoglobulin gene. Chimeric antibodies, humanized antibodies, and human antibodies have low immunogenicity in humans and are useful in using antibodies in medical preparations and the like. Therefore, when the antibody of the present invention is used for medical preparations, monoclonal antibodies are preferable, and humanized antibodies or human antibodies are particularly preferable.
　本発明の抗体は、免疫グロブリンタンパク質全長であっても、抗原と結合可能な部位を含むそれらの断片（フラグメント）であってもよい。また、抗体を含む組成物、例えば抗血清などであってもよい。 The antibody of the present invention may be a full-length immunoglobulin protein or a fragment thereof containing a site capable of binding to an antigen. It may also be a composition containing an antibody, such as antiserum.
　本発明の抗体は、抗原を免疫動物に投与することで作製することができる。抗原としては、配列番号１に示すアミノ酸配列を含むペプチドを用いることができる。抗原を免疫動物に投与する場合にあっては、ペプチドは分子量が小さく、免疫応答を十分に起こせない場合があるため、ペプチドを適当なタンパク質（例えば、アルブミン、サイログロブリン）に結合させて抗原として用いてもよい。好ましくは、抗原は配列番号１に示すアミノ酸配列を付加したサイログロブリンである。 The antibody of the present invention can be prepared by administering an antigen to an immunized animal. As the antigen, a peptide comprising the amino acid sequence shown in SEQ ID NO: 1 can be used. When an antigen is administered to an immunized animal, the peptide has a small molecular weight and may not cause an immune response sufficiently. Therefore, the peptide is bound to an appropriate protein (eg, albumin, thyroglobulin) and used as an antigen. May be. Preferably, the antigen is thyroglobulin added with the amino acid sequence shown in SEQ ID NO: 1.
　あるいは、多様な抗体またはその断片を産出しうる細胞またはファージなどのライブラリから、抗原と結合する抗体またはその断片を産出するものを探索することで、本発明の抗体を入手することもできる。この場合、抗原としては、配列番号１に示すアミノ酸配列の全長または６アミノ酸以上の一部を含むペプチドを用いることができる。好ましくは、配列番号１に示すアミノ酸配列のペプチドである。 Alternatively, the antibody of the present invention can also be obtained by searching for an antibody that binds to an antigen or a fragment thereof from a library such as a cell or phage that can produce various antibodies or fragments thereof. In this case, as the antigen, a peptide comprising the full length of the amino acid sequence shown in SEQ ID NO: 1 or a part of 6 amino acids or more can be used. Preferably, it is a peptide having the amino acid sequence shown in SEQ ID NO: 1.
　抗原として用いるペプチドの入手方法は、特に制限されず公知の方法によることができ、例えば天然由来、化学合成、無細胞系合成、組換えペプチドであってもよい。 The method for obtaining a peptide used as an antigen is not particularly limited, and can be a known method. For example, it may be naturally derived, chemically synthesized, cell-free synthesized or recombinant peptide.
　一般的な抗体の作製方法については、例えば、Harlowらの「Using Antibodies : A laboratory manual」(Cold Spring Harbor Laboratory Press, New York(1998))などにも記載されているが、以下、ポリクローナル抗体、モノクローナル抗体、およびヒト化抗体の各抗体の作製方法について簡単に説明する。 A general method for producing an antibody is described in, for example, Harlow et al., `` Using Antibodies: A laboratory manual '' (Cold Spring Harbor Laboratory Press, New York (1998)). A method for producing monoclonal antibodies and humanized antibodies will be briefly described.
　（１）ポリクローナル抗体
　ポリクローナル抗体の作製に際しては、免疫動物（例えば、マウス、ラット、ウサギ、ニワトリ）に、通常の方法、例えば、免原溶液を等量のフロイントの完全アジュバント又は不完全アジュバントと乳化混合したものを接種（初回免疫）し、以後２～４週間の間隔で数回免疫することによって行うことができる。その後、免疫した動物から血液を採取し、ポリクローナル抗体を含む抗血清を調製する。その後、ＤＥＡＥイオン交換クロマトグラフィー又はプロテインＧアフィニティークロマトグラフィー等により、抗血清からポリクローナル抗体を精製することができる。 (1) Polyclonal antibody In producing a polyclonal antibody, an immunized animal (for example, mouse, rat, rabbit, chicken) is emulsified in a usual manner, for example, an immunogen solution is emulsified with an equal volume of Freund's complete or incomplete adjuvant. The mixture can be inoculated (primary immunization) and then immunized several times at intervals of 2 to 4 weeks. Thereafter, blood is collected from the immunized animal, and antiserum containing a polyclonal antibody is prepared. Thereafter, the polyclonal antibody can be purified from the antiserum by DEAE ion exchange chromatography or protein G affinity chromatography.
　（２）モノクローナル抗体
　モノクローナル抗体は、モノクローナル抗体を産生するハイブリドーマ（モノクローナル抗体産生細胞）を分離することで作製できる。または、ファージディスプレイ法、すなわち、多様な抗体または抗体断片を発現するファージライブラリより、抗原に対して特異的に結合する抗体または抗体断片を探索する方法によって作製することもできる。 (2) Monoclonal antibody A monoclonal antibody can be prepared by isolating a hybridoma (monoclonal antibody-producing cell) that produces a monoclonal antibody. Alternatively, it can also be prepared by a phage display method, that is, a method of searching for an antibody or antibody fragment that specifically binds to an antigen from a phage library that expresses various antibodies or antibody fragments.
　ハイブリドーマを分離する方法は、Kohlerら, Nature, 256: 495 (1975)などに記載されており公知であるが、以下に簡単に説明する。最終免疫して数日後の免疫動物（例えば、マウス）から脾臓を無菌的に摘出し、脾臓から脾臓細胞を調製する。脾臓細胞は、ミエローマ細胞（骨髄腫細胞）とともに、細胞融合工程に用いる。ミエローマ細胞としては例えば、ＮＳ－１、Ｐ３Ｕ１、ＳＰ２／０などを使用することができ、細胞融合の方法としては、例えば融合促進剤（ポリエチレングリコールなど）を含む培地中で、脾臓細胞とミエローマ細胞とを、常法の混合比率（約１／５～１／１０程度の割合）で混合することにより行うことができる。融合後、選択用培地（例えば、ＨＡＴ培地）を用いて、ハイブリドーマのみを増殖させる。それらの内、培養上清中に目的のモノクローナル抗体を分泌しているハイブリドーマは、例えば、培養上清と配列番号１のアミノ酸配列のペプチドとの反応性を酵素結合免疫測定法（ＥＬＩＳＡ）でスクリーニングすることによって確認し、選択することができる。 The method for separating hybridomas is described in Kohler et al., Nature, 256: 495 (1975), etc., but is briefly described below. The spleen is aseptically removed from the immunized animal (eg, mouse) several days after the final immunization, and spleen cells are prepared from the spleen. Spleen cells are used in the cell fusion step together with myeloma cells (myeloma cells). For example, NS-1, P3U1, SP2 / 0, etc. can be used as myeloma cells. As a method for cell fusion, for example, spleen cells and myeloma cells in a medium containing a fusion promoter (polyethylene glycol, etc.) can be used. Can be carried out by mixing at a conventional mixing ratio (ratio of about 1/5 to 1/10). After the fusion, only the hybridoma is grown using a selection medium (for example, HAT medium). Among them, the hybridoma secreting the target monoclonal antibody in the culture supernatant is screened, for example, by the enzyme-linked immunoassay (ELISA) for the reactivity between the culture supernatant and the peptide having the amino acid sequence of SEQ ID NO: 1. You can confirm and choose by doing.
　ハイブリドーマの産生するモノクローナル抗体は、これを培養することにより、容易に調製することができる。培養は適切な培地中（例えば、１０％ウシ胎児血清を追加したダルベッコ変法イーグル培地（ＤＭＥＭ））で、あるいは生体内（例えば、マウスの腹腔中）で行うことができる。培養上清、あるいは腹水などを出発材料として、抗体を精製することもできる。抗体の精製には、タンパク質の精製に一般的な方法、例えば硫安塩析、イオン交換クロマトグラフィー、ゲル濾過クロマトグラフィー、プロテインＡもしくはプロテインＧ結合ポリマー等を用いるアフィニティークロマトグラフィー、または透析等の方法を適宜組み合わせて用いることができる。 Monoclonal antibodies produced by hybridomas can be easily prepared by culturing them. The culture can be performed in an appropriate medium (for example, Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum) or in vivo (for example, in the peritoneal cavity of a mouse). The antibody can also be purified using the culture supernatant or ascites as a starting material. For antibody purification, a general method for protein purification, such as ammonium sulfate salting-out, ion exchange chromatography, gel filtration chromatography, affinity chromatography using protein A or protein G binding polymer, or dialysis, is used. They can be used in appropriate combinations.
　ファージディスプレイ法は、Clacksonら, Nature 352:624-8(1991)に記載され公知であるが、以下に簡単に説明する。あらかじめ作製した多様な抗体の抗原認識部位を提示するファージライブラリから、配列表１に示すアミノ酸配列のペプチドと結合する抗原認識部位を提示するファージを選択する。選択されたファージが提示する抗原認識部位をそのまま用いても良いし、遺伝子工学的手法によって完全長の抗体を産出可能なＣＨＯ細胞などに組み込むことで、完全長の抗体を得ることもできる。 The phage display method is known and described in Clackson et al., Nature 352: 624-8 (1991), but is briefly described below. From a phage library that displays antigen recognition sites of various antibodies prepared in advance, a phage that displays an antigen recognition site that binds to a peptide having the amino acid sequence shown in Sequence Listing 1 is selected. The antigen recognition site displayed by the selected phage may be used as it is, or a full-length antibody can be obtained by incorporating the full-length antibody into a CHO cell capable of producing the full-length antibody by a genetic engineering technique.
　（３）ヒト化抗体、ヒト抗体
　ヒト化抗体とは、抗体タンパク質のうち、抗原認識のために必要最小限な領域（相補性決定領域、complementarity-determining region:ＣＤＲ）以外がヒト由来のものである。ヒト化抗体の製造方法は、例えば、ＥＰ０２３９４００、ＵＳ５５８５０８９号などに記載され公知である。 (3) Humanized antibody, human antibody A humanized antibody is an antibody protein whose origin is other than the minimum necessary region for antigen recognition (complementarity-determining region: CDR). is there. A method for producing a humanized antibody is described in, for example, EP0239400, US5585089, and the like.
　ヒト抗体は、完全なヒト由来の免疫グロブリン遺伝子を有するヒト抗体産生マウスから得ることができる。ヒト抗体産生マウスについては、ＷＯ９７／０７６７１などに記載され公知である。 A human antibody can be obtained from a human antibody-producing mouse having a complete human-derived immunoglobulin gene. Human antibody-producing mice are known and described in WO 97/07671.
　本発明の抗体がモノクローナル抗体である場合、後述の実施例で得たハイブリドーマ（受託者が付した識別のための表示：CA4.4A Hybridoma 11A1）が産生するモノクローナル抗体が好適な例として挙げられる。該ハイブリドーマは、２０１１年４月２７日（原寄託日）に、日本国千葉県木更津市かずさ鎌足２－５－８に住所を有する独立行政法人製品評価技術基盤機構　特許微生物寄託センターに受託され、２０１１年５月３１日に原寄託よりブダペスト条約に基づく寄託への移管請求が受領されたハイブリドーマである。その受託番号は、NITE BP-1090である。換言すると、本発明の抗体がモノクローナル抗体である場合、好適な例として受託番号：NITE BP-1090で特定されるハイブリドーマが産生するモノクローナル抗体が挙げられるが、これに限定されるものではない。 When the antibody of the present invention is a monoclonal antibody, a preferred example is a monoclonal antibody produced by the hybridoma obtained in the examples described later (indication for identification given by the trustee: CA4.4A Hybridoma 11A1). The hybridoma was commissioned on April 27, 2011 (original deposit date) to the Patent Microbiology Depositary Center, National Institute of Product Evaluation Technology, which has an address in 2-5-8, Kazusa Kamashita, Kisarazu City, Chiba Prefecture, Japan. The hybridoma received a request for transfer from the original deposit to the deposit under the Budapest Treaty on May 31, 2011. The accession number is NITE BP-1090. In other words, when the antibody of the present invention is a monoclonal antibody, a preferred example includes a monoclonal antibody produced by a hybridoma specified by the accession number: NITE BP-1090, but is not limited thereto.
　なお、該ハイブリドーマは、配列番号１に示すアミノ酸配列を有するポリペプチドを免疫したマウスの脾臓細胞から作製された、ＩｇＧサブタイプのモノクローナル抗体を産生するハイブリドーマである。 The hybridoma is a hybridoma that produces an IgG subtype monoclonal antibody prepared from spleen cells of a mouse immunized with a polypeptide having the amino acid sequence shown in SEQ ID NO: 1.
　本発明の抗体は、配列番号１に示すアミノ酸配列の領域と特異的に結合し、ヒトＣ４．４Ａタンパク質を特異的に検出することができる。その用途は限定されないが、癌患者の予後判定（例えば、癌再発のリスクに基づく予後、特に血行性転移に起因する癌再発のリスクに基づく予後の判定）や医薬製剤（例えば、癌再発リスクの診断薬、特に血行性転移に起因する癌再発リスクの診断薬）などに用いることができる。癌患者の予後判定については、下記に詳述する。本発明はまた、上記抗体の癌患者の予後を判定するための使用、上記抗体の医薬製剤の製造のための使用をも提供するものである。 The antibody of the present invention specifically binds to the amino acid sequence region shown in SEQ ID NO: 1 and can specifically detect human C4.4A protein. Although its use is not limited, the prognosis of cancer patients (for example, prognosis based on the risk of cancer recurrence, particularly the prognosis based on the risk of cancer recurrence due to hematogenous metastasis) and pharmaceutical preparations (eg, cancer recurrence risk) It can be used for diagnostic agents, particularly diagnostic agents for cancer recurrence risk due to hematogenous metastasis). The prognosis determination for cancer patients is described in detail below. The present invention also provides the use of the antibody for determining the prognosis of cancer patients and the use of the antibody for the production of a pharmaceutical preparation.
　２．判定方法
　本発明は、癌患者の予後を簡便かつ信頼性よく判定する方法を提供する。本発明の好ましい態様の１つにおいて、判定される癌患者の予後は、癌再発に基づく予後であり、特に血行性転移に起因する癌再発に基づく予後である。本発明はまた、癌再発のリスク、特に血行性転移に起因する癌再発リスクの判定方法でもある。 2. Determination Method The present invention provides a method for easily and reliably determining the prognosis of a cancer patient. In one preferred embodiment of the present invention, the prognosis of a cancer patient to be determined is a prognosis based on cancer recurrence, particularly a prognosis based on cancer recurrence due to hematogenous metastasis. The present invention is also a method for determining the risk of cancer recurrence, particularly the risk of cancer recurrence due to hematogenous metastasis.
　ここで、本発明の方法が対象とする癌患者が患う癌は、その種類は限定されるものではなく、あらゆる種類の癌対象となり得る。特に、例えば、胃、大腸、肺、肝、前立腺、膵、食道、膀胱、胆嚢・胆管、***、子宮、甲状腺、卵巣等における固形癌が対象として好ましく、胃、大腸、食道などの消化器系の癌が特に好ましい。精度高い判定が可能な大腸癌が、対象としてより好ましい。なお、「大腸癌」とは、盲腸、結腸（上行結腸、横行結腸、下行結腸、Ｓ状結腸）、直腸Ｓ状部、直腸（上部直腸、下部直腸）、肛門管など、大腸のあらゆる部位の癌を含む。 Here, the type of cancer affected by the cancer patient targeted by the method of the present invention is not limited, and can be any type of cancer target. In particular, for example, solid cancer in the stomach, large intestine, lung, liver, prostate, pancreas, esophagus, bladder, gallbladder / bile duct, breast, uterus, thyroid gland, ovary, etc. is preferred, and digestive system such as stomach, large intestine, esophagus Especially preferred is cancer. Colorectal cancer capable of highly accurate determination is more preferable as a target. “Colon cancer” refers to any part of the large intestine, such as the cecum, colon (ascending colon, transverse colon, descending colon, sigmoid colon), rectal sigmoid, rectum (upper rectum, lower rectum), and anal canal. Including cancer.
　本発明の方法が対象とする癌患者として、予後の判定の必要性が高いとの観点から、ＴＭＮ分類に基づく、ステージＩＩ期およびステージＩＩＩ期の癌患者が特に好ましい。ＴＭＮ分類とは、国際対がん連合（ＵＩＣＣ）によって定められた、公知の癌の進行度分類の指標である。当業者（例えば、医師。）であれば、癌患者のＴＭＮ分類に基づく癌の進行度を診断することができる。ＴＭＮ分類においては、癌の原発巣の大きさ（Tumor）、リンパ節転移の有無および個数（Node）、および遠隔転移の（Metastasis）遠隔転移の有無（Metastasis）に基づき、癌の進行がステージＩ～ＩＶに分類される。 As cancer patients targeted by the method of the present invention, stage II and stage III cancer patients based on the TMN classification are particularly preferable from the viewpoint of high prognosis determination. The TMN classification is a known index of cancer progression classification determined by the International Union for Cancer (UICC). A person skilled in the art (for example, a doctor) can diagnose the degree of cancer progression based on the TMN classification of cancer patients. In TMN classification, the progression of cancer is stage I based on the size of the primary tumor (Tumor), the presence and number of lymph node metastases (Node), and the presence of metastasis (Metastasis). It is classified into ~ IV.
　一般に、癌（例えば、大腸癌）の進行度がステージＩＩ期以上、特にステージＩＩ期およびＩＩＩ期である患者においては、癌（腫瘍）を外科的手術により除去する治療が施される。従って、本発明が対象とする癌患者は、好ましくは外科的手術などの治療処置が既に施された患者であるが、これに限定されるものではない。 Generally, in patients whose stage of progression of cancer (for example, colorectal cancer) is stage II or higher, particularly stage II and stage III, treatment for removing the cancer (tumor) by surgical operation is performed. Accordingly, the cancer patient targeted by the present invention is preferably a patient who has already undergone a therapeutic treatment such as a surgical operation, but is not limited thereto.
　また、癌患者の予後とは、ある時点、例えば癌診断時または治療時、から見て、癌の将来の状態についての予測を指し、癌の病状進行・再発・転移のリスク（可能性）が高いときは予後不良、癌の病状進行・再発・転移のリスク（可能性）が高くないときは予後良好という。 In addition, the prognosis of cancer patients refers to the prediction of the future state of cancer from a certain point of time, for example, at the time of diagnosis or treatment of cancer, and the risk (possibility) of cancer progression, recurrence, and metastasis. When the risk is high, the prognosis is poor. When the risk (probability) of cancer progression, recurrence, or metastasis is not high, the prognosis is good.
　なお、「癌再発のリスクがある」とは、特に限定されるものではないが、例えば大腸癌の進行度ステージＩ期の患者の場合は約１％（粘膜下層まで浸潤した場合）もしくは約６％（筋層以降へ浸潤した場合）、ステージＩＩ期の場合は約１３％、ステージＩＩＩ期の場合は約３０％など、統計的に知られる再発率以上の確率で癌再発のおそれがある状態を指すことができる。 “There is a risk of cancer recurrence” is not particularly limited, but for example, about 1% (when infiltrating to the submucosa) or about 6 in the case of stage I colorectal cancer patients There is a risk of cancer recurrence with a probability greater than the statistically known recurrence rate, such as% (when infiltrating into the muscle layer or later), about 13% for stage II, about 30% for stage III Can be pointed to.
　予測の指標については特定されないが、例えば全生存率（ｏｖｅｒａｌｌ　ｓｕｒｖｉｖａｌ；ＯＳ）や無病生存率（ｄｉｓｅａｓｅ ｆｒｅｅ　ｓｕｒｖｉｖａｌ；ＤＦＳ）が好適である。ここで、「全生存率」とは、一般に、観察症例のうち、特定の観察開始時点から特定の期間経過の後に生存していることが確認できる割合を意味する。「無病生存率」とは、一般に、観察症例のうち、観察開始時点から特定の期間経過の後に癌の再発なく生存していることが確認できる割合を意味する。 Although the prediction index is not specified, for example, overall survival (OS) and disease-free survival (DFS) are preferable. Here, the “total survival rate” generally means the proportion of the observed cases that can be confirmed to be alive after a specific period from the specific observation start time. “Disease-free survival rate” generally means the proportion of observed cases that can be confirmed to be alive without recurrence of cancer after a specific period from the start of observation.
　本発明の判定方法は、下記（１）～（３）の工程からなる：
（１）癌患者から採取した癌組織において、前記抗体でヒトＣ４．４Ａタンパク質の発現を検出する工程、
（２）前記工程（１）の検出結果に基づいて、癌浸潤先進部の細胞において、細胞膜と細胞質との間でヒトＣ４．４Ａタンパク質の局在を比較し、かつ癌浸潤先進部と癌病巣の表層部または中層部との間で、ヒトＣ４．４Ａタンパク質の発現量を比較する工程、および
（３）前記工程（２）の比較に基づいて、前記ヒトＣ４．４Ａタンパク質が前記癌浸潤先進部の細胞の細胞膜上に集積し、かつ前記ヒトＣ４．４Ａタンパク質の発現量が前記癌浸潤先進部の細胞において、前記癌病巣の表層部または中層部と比べて亢進している場合は、予後不良と判定する工程。 The determination method of the present invention comprises the following steps (1) to (3):
(1) detecting the expression of human C4.4A protein with the antibody in cancer tissue collected from a cancer patient;
(2) Based on the detection result of the step (1), the localization of the human C4.4A protein is compared between the cell membrane and the cytoplasm in the cells of the advanced cancer invasion, and the advanced cancer invasion and the cancer lesion are compared. Comparing the expression level of the human C4.4A protein with the surface layer portion or the middle layer portion of (3), and (3) based on the comparison in the step (2), the human C4.4A protein is Prognosis when the expression level of the human C4.4A protein is accumulated on the cell membrane of some cells and is increased in the cells in the cancer invasion advanced portion as compared with the surface layer portion or middle layer portion of the cancer lesion. The process of determining that it is defective.
　上記の（１）～（３）の工程は、下記のように換言することもできる。
（１）癌患者から採取した癌組織において、前記抗体を用いて免疫染色を行なう工程、
（２）前記工程（１）の免疫染色に基づいて、癌浸潤先進部の細胞において、細胞膜と細胞質との間でヒトＣ４．４Ａタンパク質の染色強度を比較し、かつ癌浸潤先進部と癌病巣の表層部または中層部との間で、ヒトＣ４．４Ａタンパク質の染色強度を比較する工程、および
（３）前記工程（２）の染色強度の比較に基づいて、前記ヒトＣ４．４Ａタンパク質の染色強度が、前記癌浸潤先進部の細胞の細胞膜において、前記癌浸潤先進部の細胞の細胞質と比べて亢進、かつ前記ヒトＣ４．４Ａタンパク質の染色強度が前記癌浸潤先進部の細胞において、前記癌病巣の表層部または中層部と比べて亢進している場合は、予後不良と判定する工程。さらに、上記の（１）～（３）の工程は、下記の通り換言することもできる。
（１）癌患者から採取した癌組織を、前記抗体でヒトＣ４．４Ａタンパク質について免疫測定を行う工程、
（２）前記工程（１）の免疫測定の結果について、癌浸潤先進部と癌病巣の表層部または中層部との間で、測定結果を比較する工程、および
（３）前記工程（２）の測定結果の比較に基づいて、前記ヒトＣ４．４Ａタンパク質が前記癌浸潤先進部の細胞の細胞膜上に集積し、かつ前記ヒトＣ４．４Ａタンパク質の発現量が前記癌浸潤先進部の細胞において前記癌浸潤先進部と癌病巣の表層部または中層部の細胞と比べて亢進している場合は、予後不良と判定する工程。 The above steps (1) to (3) can also be described as follows.
(1) A step of performing immunostaining using the antibody in a cancer tissue collected from a cancer patient,
(2) Based on the immunostaining in the above step (1), the staining intensity of human C4.4A protein is compared between the cell membrane and the cytoplasm in the cells of the advanced cancer invasion, and the advanced cancer invasion and the cancer lesion are compared. Comparing the staining intensity of the human C4.4A protein with the surface layer portion or the middle layer portion of the above, and (3) staining of the human C4.4A protein based on the comparison of the staining intensity of the step (2). Intensity is enhanced in the cell membrane of the advanced cancer invasion cell compared to the cytoplasm of the advanced cancer invasion cell, and the staining intensity of the human C4.4A protein is increased in the advanced cancer invasion cell. A step of determining that the prognosis is poor when the lesion is enhanced compared to the surface layer or middle layer of the lesion. Further, the above steps (1) to (3) can be expressed in other words as follows.
(1) A step of immunoassaying a cancer tissue collected from a cancer patient with respect to the human C4.4A protein with the antibody,
(2) About the result of the immunoassay in the step (1), a step of comparing the measurement result between the advanced portion of cancer invasion and the surface layer portion or middle layer portion of the cancer lesion, and (3) of the step (2) Based on the comparison of the measurement results, the human C4.4A protein is accumulated on the cell membrane of the cancer invasion advanced portion cell, and the expression level of the human C4.4A protein is increased in the cancer invasion advanced portion cell. A step of determining a poor prognosis when the invasion advanced portion and cells in the surface layer or middle layer of the cancer lesion are enhanced.
 以下、各工程について説明する。 Hereinafter, each step will be described.
　（１）癌患者から採取した癌組織において、前記抗体でヒトＣ４．４Ａタンパク質の発現を検出する工程、について。 (1) Regarding the step of detecting the expression of human C4.4A protein with the antibody in cancer tissue collected from cancer patients.
　癌患者から採取した癌組織においてヒトＣ４．４Ａタンパク質の発現を検出、すなわち前記抗体を用いて免疫染色（免疫組織化学）を行なう方法は、公知の手法を用いて行なうことができる。 A method for detecting the expression of human C4.4A protein in a cancer tissue collected from a cancer patient, that is, performing immunostaining (immunohistochemistry) using the antibody can be performed using a known method.
　例えば、免疫染色（免疫組織化学）を行う場合にあっては、組織検体を固定する場合もある。具体的には、検体をホルマリン溶液による固定、およびエタノール溶液による脱水を行うことが好適であるが、これに限定されない。さらに、固定化された検体は、パラフィンに包埋し、最終的にパラフィンブロックから試料切片を調製することができる。 For example, when performing immunostaining (immunohistochemistry), a tissue specimen may be fixed. Specifically, the specimen is preferably fixed with a formalin solution and dehydrated with an ethanol solution, but is not limited thereto. Furthermore, the immobilized specimen can be embedded in paraffin, and finally a sample section can be prepared from the paraffin block.
　続いて、検体と抗体を接触させるが、この方法も公知である。例えば、０．１～１００ｍｇ／ｍｌで抗体を含むＰＢＳ溶液と検体を接触させ、温度４～３７℃にて、６０分～一晩反応させてもよい。 Subsequently, the specimen and the antibody are brought into contact, and this method is also known. For example, the sample may be brought into contact with a PBS solution containing an antibody at 0.1 to 100 mg / ml and reacted at a temperature of 4 to 37 ° C. for 60 minutes to overnight.
　続いて、抗体とヒトＣ４．４Ａタンパク質の結合の可視化を行なうが、この方法も公知である。方法の簡便性を考慮すると、標識化された二次抗体を用いることが好ましい。可視化を行うにあたり標識に用いた蛍光を検出する場合などにおいては、ＣＣＤカメラを備えた蛍光顕微鏡システムによって可視化を行うことができる。あるいは、現像写真を得ることもできる。 Subsequently, the binding between the antibody and the human C4.4A protein is visualized, and this method is also known. Considering the simplicity of the method, it is preferable to use a labeled secondary antibody. In the case of detecting the fluorescence used for the label for visualization, the visualization can be performed by a fluorescence microscope system equipped with a CCD camera. Alternatively, a developed photograph can be obtained.
　かくして、工程（１）により、ヒトＣ４．４Ａタンパク質の発現が検出される。 Thus, the expression of human C4.4A protein is detected by step (1).
　（２）前記工程（１）の検出結果に基づいて、癌浸潤先進部の細胞において、細胞膜と細胞質との間でヒトＣ４．４Ａタンパク質の局在を比較し、かつ癌浸潤先進部と癌病巣の表層部または中層部との間で、ヒトＣ４．４Ａタンパク質の局在および発現量を比較する工程、について。 (2) Based on the detection result of the step (1), the localization of the human C4.4A protein is compared between the cell membrane and the cytoplasm in the cells of the advanced cancer invasion, and the advanced cancer invasion and the cancer lesion are compared. Comparing the localization and expression level of the human C4.4A protein between the surface layer and the middle layer.
　工程（１）の結果を評価する。評価は、ヒトＣ４．４Ａタンパク質が検出される細胞内の箇所および検出される相対物質量について行う。具体的には、工程（１）で得られるヒトＣ４．４Ａタンパク質の発現パターンに基づき、ヒトＣ４．４Ａタンパク質の局在および発現量を比較する。換言すると、工程（２）では、前記工程（１）の免疫染色に基づいて、癌浸潤先進部の細胞において、細胞膜と細胞質との間でヒトＣ４．４Ａタンパク質の染色強度を比較し、かつ癌浸潤先進部と癌病巣の表層部または中層部との間で、ヒトＣ４．４Ａタンパク質の染色強度を比較する Evaluate the result of step (1). Evaluation is performed about the location in the cell in which human C4.4A protein is detected, and the relative substance amount detected. Specifically, the localization and expression level of human C4.4A protein are compared based on the expression pattern of human C4.4A protein obtained in step (1). In other words, in the step (2), the staining intensity of the human C4.4A protein is compared between the cell membrane and the cytoplasm in the cells in the advanced part of cancer invasion based on the immunostaining in the step (1), and the cancer Compare the staining intensity of human C4.4A protein between the advanced invasion and the surface layer or middle layer of the cancer lesion
　ヒトＣ４．４Ａタンパク質の局在および発現量を比較するための評価方法、すなわちヒトＣ４．４Ａタンパク質の染色強度の評価方法は、当業者が適宜選択すればよい。 An evaluation method for comparing the localization and expression level of human C4.4A protein, that is, a method for evaluating the staining intensity of human C4.4A protein may be appropriately selected by those skilled in the art.
　ここで、「癌浸潤先進部」とは、病理学的所見により癌が癌化した組織の表層部から深部に向かって成長する最深部を指し、「癌病巣の表層部または中層部（中間部）」は、先進部以外の部位全体を指す。癌浸潤先進部の細胞の細胞膜および細胞質は、当業者が通常の手段（例えば、形態に基づく識別）により識別することができる。 Here, the “advanced portion of cancer invasion” refers to the deepest portion that grows from the surface layer portion to the deep portion of the tissue in which cancer has become cancerous by pathological findings, and refers to the “surface layer portion or middle layer portion (intermediate portion of the cancer lesion). ")" Refers to the entire region other than the advanced part. The cell membrane and cytoplasm of cells in advanced cancer invasion can be identified by those skilled in the art by conventional means (eg, identification based on morphology).
　しばしば、消化器系などの組織においては、癌は器官の表層部（例えば、上皮、粘膜など）で生じる。当初癌化した箇所が原病巣である。その後、生じた癌は、細胞数を増大させながら、周辺部位へ向かって成長（すなわち、浸潤。）をしていく。周辺とは、例えば大腸においては、表層に近い順に、粘膜下層、筋層などを指す。成長をする癌病巣部において、粘膜下層、筋層などの癌化していない周辺部位と接する、癌病巣部の最深部を「癌浸潤先進部」という。さらに癌が成長をしていくと、以前には周辺部位と接していた部位が、もはや最深部ではなくなると、「中層部（中間部）」となる。 Often, in tissues such as the digestive system, cancer occurs in the surface layer of the organ (eg, epithelium, mucous membrane, etc.). The original cancerous part is the original lesion. Thereafter, the resulting cancer grows (ie, infiltrates) toward the peripheral site while increasing the number of cells. For example, in the large intestine, the periphery refers to the submucosal layer, the muscle layer, and the like in order from the surface layer. In the growing cancer lesion, the deepest portion of the cancer lesion that contacts non-cancerous peripheral sites such as the submucosa and muscle layers is referred to as the “cancer invasion advanced portion”. As the cancer further grows, when the site that was previously in contact with the surrounding site is no longer the deepest part, it becomes the “middle layer (intermediate part)”.
　比較に際して、「癌病巣の表層部または中層部」としては、表層部および中層部のいずれであってもよい。前述の通り、癌浸潤先進部と中層部とが位置関係が近い場合が多い。そのため、実用性の観点から、癌浸潤先進部と、癌病巣の中層部との間で比較を行なうことが好ましい。 For comparison, the “surface layer portion or middle layer portion of the cancer lesion” may be either the surface layer portion or the middle layer portion. As described above, the cancer invasion advanced portion and the middle layer are often close in positional relationship. Therefore, from the viewpoint of practicality, it is preferable to make a comparison between the cancer invasion advanced part and the middle part of the cancer lesion.
　工程（１）の結果を、癌浸潤先進部の細胞において、細胞膜と細胞質との間、および、癌浸潤先進部と癌病巣の表層部または中層部との間で比較すると、比較結果は下記の４つのパターンに分類できる（後述の実施例２および図４参照）：
（Ａ）　ヒトＣ４．４Ａタンパク質が癌浸潤先進部の細胞の細胞膜上に集積し、かつヒトＣ４．４Ａタンパク質の発現量が癌浸潤先進部の細胞において、癌病巣の表層部または中層部と比べて亢進している、
（Ｂ１）ヒトＣ４．４Ａタンパク質が癌浸潤先進部の細胞の細胞膜上に集積しているが、ヒトＣ４．４Ａタンパク質の発現量が癌浸潤先進部と癌病巣の表層部または中層部との間で同程度である
（Ｂ２）ヒトＣ４．４Ａタンパク質が癌浸潤先進部の細胞の細胞膜上に集積していないが、ヒトＣ４．４Ａタンパク質の発現量が癌浸潤先進部の細胞において、癌病巣の表層部または中層部と比べて亢進している、
（Ｃ）　ヒトＣ４．４Ａタンパク質が癌浸潤先進部の細胞の細胞膜上に集積しておらず、ヒトＣ４．４Ａタンパク質の発現量が癌浸潤先進部と癌病巣の表層部または中層部との間で同程度である。 When the results of step (1) are compared between the cell membrane and the cytoplasm, and between the cancer invasion advanced portion and the surface layer portion or middle layer portion of the cancer lesion in the cells of the cancer invasion advanced portion, the comparison results are as follows. It can be classified into four patterns (see Example 2 and FIG. 4 described later):
(A) Human C4.4A protein is accumulated on the cell membrane of cells in advanced cancer invasion, and the expression level of human C4.4A protein is higher than that in the surface layer or middle layer of cancer lesions in cells in advanced cancer invasion Is increasing,
(B1) Human C4.4A protein is accumulated on the cell membrane of cells in advanced cancer invasion, but the expression level of human C4.4A protein is between the advanced cancer invasion and the surface layer or middle layer of the cancer lesion. (B2) Although human C4.4A protein is not accumulated on the cell membrane of cells in advanced cancer invasion, the expression level of human C4.4A protein is Increased compared to the surface layer or middle layer,
(C) Human C4.4A protein is not accumulated on the cell membrane of cells in advanced cancer invasion, and the expression level of human C4.4A protein is between the advanced part of cancer invasion and the surface layer or middle layer of the cancer lesion. It is about the same.
　比較結果は、以下のように換言することができる。
（Ａ）　ヒトＣ４．４Ａタンパク質の染色強度が、癌浸潤先進部の細胞の細胞膜において、癌浸潤先進部の細胞の細胞質と比べて亢進、かつヒトＣ４．４Ａタンパク質の染色強度が、癌浸潤先進部の細胞において、癌病巣の表層部または中層部と比べて亢進している、
（Ｂ１）ヒトＣ４．４Ａタンパク質の染色強度が、癌浸潤先進部の細胞の細胞膜上において、癌浸潤先進部の細胞の細胞質と比べて亢進しているが、ヒトＣ４．４Ａタンパク質の染色強度が、癌浸潤先進部と癌病巣の表層部または中層部との間で同程度である
（Ｂ２）ヒトＣ４．４Ａタンパク質の染色強度が、癌浸潤先進部の細胞の細胞膜上において癌浸潤先進部の細胞の細胞質と比べて亢進していないが、ヒトＣ４．４Ａタンパク質の染色強度が、癌浸潤先進部の細胞において、癌病巣の表層部または中層部と比べて亢進している、
（Ｃ）ヒトＣ４．４Ａタンパク質の染色強度が、癌浸潤先進部の細胞の細胞膜上において、癌浸潤先進部の細胞の細胞質と比べて亢進しておらず、ヒトＣ４．４Ａタンパク質の染色強度が、癌浸潤先進部と癌病巣の表層部または中層部との間で同程度である。 The comparison result can be rephrased as follows.
(A) The staining intensity of human C4.4A protein is enhanced in the cell membrane of cells in advanced cancer invasion compared to the cytoplasm of cells in advanced cancer invasion, and the staining intensity of human C4.4A protein is advanced in cancer invasion. In some cells, compared to the surface layer or middle layer of the cancer lesion,
(B1) Although the staining intensity of human C4.4A protein is enhanced on the cell membrane of cells in advanced cancer invasion compared to the cytoplasm of cells in advanced cancer invasion, the staining intensity of human C4.4A protein is increased. (B2) The staining intensity of the human C4.4A protein is comparable between the advanced part of cancer invasion and the surface layer or middle part of the cancer lesion. Although it is not enhanced compared with the cytoplasm of the cell, the staining intensity of human C4.4A protein is enhanced in the cells in the advanced part of cancer invasion compared to the surface layer or middle layer of the cancer lesion.
(C) The staining intensity of human C4.4A protein is not enhanced on the cell membrane of cells in advanced cancer invasion compared to the cytoplasm of cells in advanced cancer invasion, and the staining intensity of human C4.4A protein is It is the same level between the advanced part of cancer invasion and the surface part or middle part of the cancer lesion.
　なお、「ヒトＣ４．４Ａタンパク質が癌浸潤先進部の細胞の細胞膜上に集積」とは、癌浸潤先進部の細胞の細胞膜において、癌浸潤先進部の細胞の細胞質と比べてヒトＣ４．４Ａタンパク質の発現量が亢進している状態を指す。 “Human C4.4A protein is accumulated on the cell membrane of cells in advanced cancer invasion” means that the human C4.4A protein is compared with the cytoplasm of cells in advanced cancer invasion in the cell membrane of advanced cancer invasion. Refers to a state in which the expression level of is increased.
　かくして、工程（１）の結果が評価され、ヒトＣ４．４Ａタンパク質の発現パターンが比較される。 Thus, the result of step (1) is evaluated and the expression patterns of human C4.4A protein are compared.
　（３）前記工程（２）の比較に基づいて、前記ヒトＣ４．４Ａタンパク質が前記癌浸潤先進部の細胞の細胞膜上に集積し、かつ前記ヒトＣ４．４Ａタンパク質の発現量が前記癌浸潤先進部の細胞において、癌病巣の表層部または中層部と比べて亢進している場合は、予後不良と判定する工程、について。 (3) Based on the comparison in the step (2), the human C4.4A protein is accumulated on the cell membrane of the cell in the cancer invasion advanced part, and the expression level of the human C4.4A protein is the cancer invasion advanced. When the cell of the part is elevated as compared with the surface layer or middle layer of the cancer lesion, the step of determining a poor prognosis.
　上記工程（２）で分類される４パターンのうち、Ａ型の発現パターンであった検体が由来した患者について、癌の予後が不良である、すなわち癌再発のリスクがある、と判定する。このことは、後述の実施例２、図６および図７において示すように、Ａ型の発現パターンであることは、悪性浸潤の指標である腫瘍蔟出（tumor budding）が高頻度に検出されることとは統計学的に有意に相関すること、ならびに５年間全生存率（ＯＳ）および５年間無病生存率（ＤＦＳ）について（Ｂ１＋Ｂ２＋Ｃ）型の発現パターンである場合と比べて統計学的に有意な予後不良が認められることに基づく。 Among the four patterns classified in the above step (2), it is determined that the patient from whom the specimen that was the expression pattern of type A is derived has a poor cancer prognosis, that is, there is a risk of cancer recurrence. As shown in Example 2, FIG. 6 and FIG. 7 described later, this is an expression pattern of type A, and tumor budding, which is an index of malignant invasion, is detected at a high frequency. Are statistically significant and statistically significant for the 5-year overall survival (OS) and 5-year disease-free survival (DFS) compared to the (B1 + B2 + C) type expression pattern Based on the fact that a poor prognosis is observed.
　予後不良と判定された患者については、アフターケア（主に、手術後のアフターケア）として、慎重な経過観察および重点的な予防的措置を行うことが好ましい。慎重な経過観察および重点的な予防的措置の具体例として、フルオロウラシル（５－ＦＵ）、レボホリナート（ＬＶ；商品名：アイソボリン、ファイザー・インコーポレーテッド）、オキサリプラチン（Ｌ－ＯＨＰ；商品名：エルプラット／Eloxatin、ヤクルト本社）などの抗癌剤を投与する等の措置が挙げられるが、これに限定されるものではない。 For patients determined to have a poor prognosis, it is preferable to perform careful follow-up and intensive preventive measures as aftercare (mainly aftercare after surgery). Specific examples of careful follow-up and focused preventive measures include fluorouracil (5-FU), levofolinate (LV; trade name: Isobolin, Pfizer Inc.), oxaliplatin (L-OHP; trade name: Elplat) / Eloxatin, Yakult Headquarters) and other measures such as administration of anti-cancer agents are included, but are not limited thereto.
　発現パターンがＢ１型またはＢ２型であった場合には、ただちに予後不良とは判定されないものの、慎重な経過観察が望ましい。 If the expression pattern is B1 or B2, it is not immediately determined that the prognosis is poor, but careful follow-up is desirable.
　Ｃ型の発現パターンで合った場合は、ただちに予後不良であるとは判定されない。適度な経過観察や予防的措置のみを行い、経過に応じた措置を行っていくことが好ましい。 If it matches with the C-type expression pattern, it is not immediately determined that the prognosis is poor. It is preferable to perform only appropriate follow-up observations and preventive measures, and take measures according to the progress.
　また、本発明の判定方法の他の公知の、あるいは将来的に見出される癌の予後判定方法と組み合わせてもよい。例えば、癌検体の病理組織学的所見（具体的には、リンパ節転移の有無などが挙げられる。）、癌患者の遺伝的要因の検出などとの組み合わせが挙げられるが、これに限定されない。 Further, the determination method of the present invention may be combined with other known or prognosis determination methods for cancers to be found in the future. Examples include, but are not limited to, a combination of histopathological findings of cancer specimens (specifically, the presence or absence of lymph node metastasis), detection of genetic factors in cancer patients, and the like.
　かくして、癌患者の予後不良が判定される。本発明の方法が、癌再発リスクの判定方法である場合は、癌再発のリスクの有無が判定される。 Thus, poor prognosis for cancer patients is determined. When the method of the present invention is a method for determining the risk of cancer recurrence, the presence or absence of the risk of cancer recurrence is determined.
　３．キット
　本発明のキットは、癌患者の予後を判定するためのキットであって、上記の抗体を含んでなるキットである。 3. Kit The kit of the present invention is a kit for determining the prognosis of a cancer patient and comprising the above-described antibody.
　また、本発明のキットには、上記抗体以外に、必要に応じて他の成分を含めることができる。他の成分は、例えば免疫測定を行うために必要な試薬または器具であってもよい。具体的には、免疫組織化学を行う際の検体固定に用いる試薬（例えば、ホルマリン、エタノール）、検出に用いる試薬（例えば、２次抗体）、使用する器具（例えば、スライドガラス、カバーカラス）、もしくはポジティブコントロール試料およびネガティブコントロール試料などが挙げられるが、これに制限されない。さらに、上記判定方法を行うための手順を書き記した書面などを含むことができる。 In addition, the kit of the present invention may contain other components as necessary in addition to the above-described antibodies. Other components may be, for example, reagents or instruments necessary to perform an immunoassay. Specifically, reagents used for specimen fixation when performing immunohistochemistry (for example, formalin, ethanol), reagents used for detection (for example, secondary antibody), instruments to be used (for example, slide glass, cover crow), Or a positive control sample, a negative control sample, etc. are mentioned, However It is not restricted to this. Furthermore, the document etc. which wrote down the procedure for performing the said determination method can be included.
　本発明のキットは、常法に従い、必要に応じて上記成分を備えることで作製することができる。 The kit of the present invention can be prepared by providing the above-mentioned components as necessary according to a conventional method.
　キットの使用形態は特に限定されないが、上記判定方法に用いることが好ましい。上記判定方法に用いた場合、判定を容易に行うことが可能となる。本発明のキットの好ましい態様において、判定される癌患者の予後は、癌再発リスクに基づく予後であり、特に血行性転移に起因する癌再発リスクに基づく予後である。 The usage form of the kit is not particularly limited, but it is preferably used in the above determination method. When used in the above determination method, the determination can be easily performed. In a preferred embodiment of the kit of the present invention, the prognosis of a cancer patient to be determined is a prognosis based on the risk of cancer recurrence, particularly a prognosis based on the risk of cancer recurrence due to hematogenous metastasis.
　４．診断薬
　本発明の癌診断薬は、上記の抗体を含んでなる。 4). Diagnostic Agent The cancer diagnostic agent of the present invention comprises the antibody described above.
　本発明の癌診断薬の好ましい態様の１つとして、癌再発リスクの診断薬が挙げられる。特に好ましい態様は、血行性転移に起因する癌再発リスクの診断薬である。 One preferred embodiment of the cancer diagnostic agent of the present invention is a diagnostic agent for cancer recurrence risk. A particularly preferred embodiment is a diagnostic agent for cancer recurrence risk due to hematogenous metastasis.
　また、本発明の診断薬には、抗体を不活性化させず、上記判定方法を行うに当たって支障がない限り、他の成分を含めることができる。具体的には、生理食塩水、緩衝液などが挙げられるが、これに限定されるものではない。 Further, the diagnostic agent of the present invention can contain other components as long as the antibody is not inactivated and there is no problem in performing the above-described determination method. Specific examples include physiological saline and buffer solution, but are not limited thereto.
　本発明の診断薬は、常法に従い、必要に応じて上記成分を備えることで調製することができる。 The diagnostic agent of the present invention can be prepared by providing the above components as necessary according to a conventional method.
　実施例
　以下に、実施例等に基づいて本発明を詳細に説明するが、本発明はこれらによって限定されるものではない。 EXAMPLES Hereinafter, the present invention will be described in detail based on examples and the like, but the present invention is not limited thereto.
　［実施例１］
ポリクローナル抗体の作製と特異性の検定
＜Ｉ．実験材料と実験方法＞
１．ポリクローナル抗体の作製
　ウサギを、標的ペプチドを結合したサイログロブリンで免疫した。標的ペプチドは、ヒトＣ４．４Ａタンパク質のＮ末端近傍の８３～９７番目のアミノ酸配列に相当する配列番号２に示すアミノ酸配列を有するペプチド、およびヒトＣ４．４Ａタンパク質のＣ末端の３０１～３１６番目のアミノ酸配列に相当する、ＧＰＩ付加シグナル配列の一部を含む配列番号１に示すアミノ酸配列を有するペプチドを用いた。（図１）。それぞれのウサギから採取した抗血清を、標的ペプチドが担体ビーズに結合されたカラムに通過させることで、ヒトＣ４．４Ａタンパク質に対して特異的に結合するＩｇＧを精製した。得られたＮ末端およびＣ末端に対する抗体を、それぞれ抗Ｃ４．４Ａ－１抗体および抗Ｃ４．４Ａ－２抗体と命名した。 [Example 1]
Preparation of polyclonal antibody and specificity test <I. Experimental materials and methods>
1. Production of polyclonal antibodies Rabbits were immunized with thyroglobulin conjugated with the target peptide. The target peptide is a peptide having the amino acid sequence shown in SEQ ID NO: 2 corresponding to the 83rd to 97th amino acid sequence near the N-terminal of the human C4.4A protein, and the 301st to 316th of the C-terminal of the human C4.4A protein. A peptide having the amino acid sequence shown in SEQ ID NO: 1 containing a part of the GPI addition signal sequence corresponding to the amino acid sequence was used. (FIG. 1). The antiserum collected from each rabbit was passed through a column in which the target peptide was bound to carrier beads, thereby purifying IgG that specifically binds to human C4.4A protein. The obtained antibodies against the N-terminus and C-terminus were named anti-C4.4A-1 antibody and anti-C4.4A-2 antibody, respectively.
２．細胞株
　ヒト大腸癌由来細胞株ＨＣＴ１１６は、米国アメリカン・セル・タイプ・コレクション（米国バージニア州マナサス）より入手した。ヒト大腸癌由来細胞株ＫＭ１２ＳＭは、金沢大学癌研究所の源利成教授から分与されたものを用いた。これらの細胞株は、１０%のウシ胎児血清（ＦＢＳ）、１００ユニット／ｍＬのペニシリン、および１００マイクログラム／ｍＬのストレプトマイシンを追加したダルベッコ変法イーグル培地（ＤＭＥＭ）中、ならびに温度３７℃および空気中の二酸化炭素濃度５％の恒温恒湿槽中にて培養を行った。 2. Cell line Human colon cancer-derived cell line HCT116 was obtained from American Cell Type Collection (Manassas, VA, USA). The human colorectal cancer-derived cell line KM12SM used was distributed from Professor Kanazawa University Cancer Research Institute. These cell lines are in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 units / mL penicillin, and 100 microgram / mL streptomycin, and at a temperature of 37 ° C. and air Culturing was performed in a constant temperature and humidity chamber having a carbon dioxide concentration of 5%.
３．大腸癌組織検体
　大腸癌組織検体は、インフォームドコンセントを行った大腸癌患者より、大阪大学外科学講座（ｎ＝１２２，１９９５～２００５）および九州大学生体防御医学研究所（ｎ＝１０８，１９９３～１９９９）における手術時に採取されたものを使用した。採取された組織検体は、緩衝ホルマリン液で一晩、４℃にて固定した後、エタノール系列で脱水処理をし、パラフィンに包埋した。 3. Colorectal cancer tissue specimens Colorectal cancer tissue specimens were obtained from Osaka University Department of Surgery (n = 122, 1995-2005) and Kyushu University Institute of Biomedical Defense (n = 108, 1993- 1999) were used at the time of surgery. The collected tissue specimens were fixed overnight in buffered formalin solution at 4 ° C., then dehydrated with an ethanol series, and embedded in paraffin.
４．ウェスタンブロット法
　ウェスタンブロット解析は常法に従って行った。以下にその概略を示す。タンパク質試料（２０マイクログラム）を、１２．５％ポリアクリルアミドゲル電気泳動で分離し、その後ポリフッ化ビニリデン（ＰＶＤＦ）メンブラン上に電気ブロッテイングした。メンブランは各々の抗体の至適濃度で１時間インキュベートした。タンパク質のバンドは、ＥＣＬウェスタンブロッティング検出システム（アマシャム社、米国ニュージャージー州）を用いて検出した。 4). Western Blot Western blot analysis was performed according to a conventional method. The outline is shown below. Protein samples (20 micrograms) were separated by 12.5% polyacrylamide gel electrophoresis and then electroblotted onto a polyvinylidene fluoride (PVDF) membrane. The membranes were incubated for 1 hour at the optimal concentration of each antibody. Protein bands were detected using an ECL Western blotting detection system (Amersham, NJ, USA).
５．免疫組織化学
　４マイクロミリメートル厚の組織切片を、パラフィン包埋ブロックより調製した。抗原賦活化処理を１０ｍＭクエン酸緩衝液中にて、温度９５℃下で４０分間行った後に、抗体染色をＶｅｔａｓｔａｉｎ　ＡＢＣ　ｐｅｒｏｘｉｄａｓｅ　ｋｉｔ（Ｖｅｃｔｏｒ　Ｌａｂｓ社、米国カリフォルニア州）を用いて行った。スライドグラスを、５０μｇ／ｍｌの抗体原液を１：２００に希釈した抗Ｃ４．４Ａ－１抗体または抗Ｃ４．４－２抗体中で、一晩４℃にてインキュベートした。非免疫ウサギ由来ＩｇＧ（Ｖｅｃｔｏｒ　Ｌａｂｏｒａｔｏｒｉｅｓ社）をネガティブコントロールとして利用し、１次抗体の代わりに用いることで、２次抗体またはＩｇＧの非特異的結合による偽陽性反応の可能性を排除した。 5. Immunohistochemistry 4 micromm thick tissue sections were prepared from paraffin embedded blocks. Antigen activation treatment was performed in a 10 mM citrate buffer solution at a temperature of 95 ° C. for 40 minutes, and then antibody staining was performed using a Betastain ABC peroxide kit (Vector Labs, California, USA). The slides were incubated overnight at 4 ° C. in anti-C4.4A-1 antibody or anti-C4.4-2 antibody diluted 1: 200 in 50 μg / ml antibody stock solution. Non-immune rabbit-derived IgG (Vector Laboratories) was used as a negative control and used in place of the primary antibody to eliminate the possibility of false positive reaction due to non-specific binding of the secondary antibody or IgG.
＜ＩＩ．実験結果＞
１．抗Ｃ４．４Ａポリクローナル抗体の反応性：ウェスタンブロット法
　作製した２種類の抗体についてウェスタンブロット解析を行ったところ、ポジティブコントロールの食道上皮検体については、両方の抗Ｃ４．４Ａ抗体は、分子量６７ｋＤａ近傍に明確なバンドを示した（図２）。しかし、大腸癌由来細胞株および大腸組織検体については、抗Ｃ４．４Ａ－２ポリクローナル抗体のみが約４０ｋＤａの位置に顕著なバンドを示し、一方で抗Ｃ４．４Ａ－１抗体はバンドを示さなかった（図２）。 <II. Experimental results>
1. Anti-C4.4A Polyclonal Antibody Reactivity: Western Blot Western blot analysis was performed on the two types of antibodies produced. As a result of positive control esophageal epithelial specimens, both anti-C4.4A antibodies had a molecular weight of around 67 kDa. A clear band was shown (Figure 2). However, for colon cancer-derived cell lines and colon tissue samples, only the anti-C4.4A-2 polyclonal antibody showed a significant band at a position of about 40 kDa, whereas the anti-C4.4A-1 antibody showed no band. (FIG. 2).
　なお、ローディングコントロールとして用いたウサギ由来の抗ヒトアクチン抗体は、シグマアルドリッチ社（米国ミズーリ州セントルイス）から購入した。 The rabbit-derived anti-human actin antibody used as a loading control was purchased from Sigma-Aldrich (St. Louis, MO, USA).
２．抗Ｃ４．４Ａポリクローナル抗体の反応性：免疫組織化学
　上記と一致した結果が、免疫組織化学によっても得られた。すなわち、両方の抗体について、基底上の扁平上皮において染色が観察された（図３Ａ、Ｂ）。大腸癌組織検体においては、抗Ｃ４．４Ａ－１抗体は何らシグナルが検出されなかったものの、抗Ｃ４．４Ａ－２抗体では染色シグナルが得られた（図３Ｃ、Ｄ）。ネガティブコントロール検定を行ったところ、非免疫ウサギ由来ＩｇＧおよび過剰量のヒトＣ４．４Ａ－２抗原ペプチドにあらかじめ吸着させた抗体の何れもが、食道上皮組織検体を染色しなかった。（データは示さない）。 2. Reactivity of anti-C4.4A polyclonal antibody: immunohistochemistry Results consistent with the above were also obtained by immunohistochemistry. That is, for both antibodies, staining was observed in the squamous epithelium on the base (FIG. 3A, B). In the colorectal cancer tissue specimen, no signal was detected with the anti-C4.4A-1 antibody, but a staining signal was obtained with the anti-C4.4A-2 antibody (FIGS. 3C and D). When a negative control assay was performed, none of the IgG pre-adsorbed to non-immune rabbit-derived IgG and an excess amount of human C4.4A-2 antigen peptide stained the esophageal epithelial tissue specimen. (Data not shown).
＜ＩＩＩ．考察＞
　このように、配列番号１に示すアミノ酸配列の領域に特異的に結合する抗Ｃ４．４Ａ－２抗体は、大腸癌由来の試料において、ヒトＣ４．４Ａタンパク質を特異的に検出することができた。一方、抗Ｃ４．４Ａ－１抗体はヒトＣ４．４Ａタンパク質の検出ができなかった。 <III. Discussion>
Thus, the anti-C4.4A-2 antibody that specifically binds to the region of the amino acid sequence shown in SEQ ID NO: 1 was able to specifically detect human C4.4A protein in a sample derived from colorectal cancer. . On the other hand, anti-C4.4A-1 antibody could not detect human C4.4A protein.
　また、ヒトＣ４．４Ａタンパク質は、配列番号３に示す全長配列の３０８番目と３０９番目とのアミノ酸残基の間で切断されて、ＧＰＩアンカーが付加されると予想されている。従って、本発明の抗体は配列番号３に示す全長配列の３０１～３０８番目のアミノ酸配列の領域を認識していると推定される。 In addition, the human C4.4A protein is predicted to be cleaved between the 308th and 309th amino acid residues of the full-length sequence shown in SEQ ID NO: 3 and a GPI anchor is added. Therefore, the antibody of the present invention is presumed to recognize the region of the 301st to 308th amino acid sequence of the full-length sequence shown in SEQ ID NO: 3.
　［実施例２］
大腸癌組織検体におけるヒトＣ４．４Ａタンパク質の検出、および生存率との相関
＜Ｉ．実験方法＞
　１．大腸癌組織検体におけるヒトＣ４．４Ａタンパク質の検出
　実施例１に準じた方法で、大腸癌組織検体におけるヒトＣ４．４Ａタンパク質の検出を、抗Ｃ４．４Ａー２ポリクローナル抗体を用いて行った。 [Example 2]
Detection of human C4.4A protein in colorectal cancer tissue specimens and correlation with survival rate <I. Experimental method>
1. Detection of human C4.4A protein in colorectal cancer tissue specimens According to the method according to Example 1, detection of human C4.4A protein in colorectal cancer tissue specimens was performed using an anti-C4.4A-2 polyclonal antibody.
　２．腫瘍蔟出の測定
　腫瘍蔟出は、腫瘍の悪性浸潤性の指標である。そこで、腫瘍蔟出のヒトＣ４．４Ａタンパク質の発現パターンと腫瘍蔟出の関係を調べるために、腫瘍蔟出の測定を行った。腫瘍蔟出の評価は、Shintoら, Dis Colon Rectum49: 1422-1430 (2006)に記載の方法に従った。以下にその概略を記す。単一の孤立した癌細胞、または４以内の癌細胞からなる細胞集団を、腫瘍蔟出と定義した。２０倍の対物レンズ下で観察したときに、最も高密度に腫瘍蔟出が観察される領域における腫瘍蔟出の出現頻度に応じて、癌組織を２つのグループに分類した。腫瘍蔟出の出現頻度が０から９である場合を低頻度蔟出とし、腫瘍蔟出の現頻度が１０以上である場合を高頻度蔟出と定義した。 2. Tumor eruption measurement Tumor eruption is an indicator of tumor malignancy. Therefore, in order to examine the relationship between the expression pattern of human C4.4A protein in tumor exudation and tumor exudation, tumor exudation was measured. Tumor exudation was evaluated according to the method described in Shinto et al., Dis Colon Rectum 49: 1422-1430 (2006). The outline is described below. A single isolated cancer cell or a cell population consisting of up to 4 cancer cells was defined as tumor enucleation. When observed under a 20 × objective lens, the cancer tissues were classified into two groups according to the appearance frequency of tumor exudation in the region where tumor exudation was observed at the highest density. A case where the appearance frequency of tumor eruption was 0 to 9 was defined as low-frequency eruption, and a case where the present frequency of tumor eruption was 10 or more was defined as high-frequency eruption.
　３．統計解析
　大腸癌組織検体におけるヒトＣ４．４Ａタンパク質の発現パターンと生存率との相関を統計解析によって評価した。統計解析は、ＳｔａｔＶｉｅｗＪ－５．０プログラム（アバカスコンセプツ社、米国カリフォルニア州バークレー）を用いて行った。大腸癌再発率の推定を行うためにはカプランマイヤー法を用い、統計的有意性の検定のためにはログランク検定を用いた。離散変数間の関連性は、カイ２乗検定を用いて評価した。平均値の比較は、マンホイットニー検定を用いて行った。スピアマンの順位相関検定を用いて、２因子間の相関関係を解析した。データは、中央値をもって表記した。Ｐ値が＜０．０５の場合を、統計的有意差があるとした。 3. Statistical analysis The correlation between human C4.4A protein expression pattern and survival rate in colorectal cancer tissue specimens was evaluated by statistical analysis. Statistical analysis was performed using the StatView J-5.0 program (Abacus Concepts, Berkeley, California, USA). The Kaplan-Meier method was used to estimate the recurrence rate of colorectal cancer, and the log rank test was used to test for statistical significance. Associations between discrete variables were evaluated using the chi-square test. Comparison of mean values was performed using Mann-Whitney test. Spearman's rank correlation test was used to analyze the correlation between the two factors. Data are expressed as medians. A case where the P value was <0.05 was considered to be statistically significant.
　＜ＩＩ．実験結果＞
　１．大腸癌組織および浸潤部位におけるヒトＣ４．４Ａタンパク質の発現パターン <II. Experimental results>
1. Expression pattern of human C4.4A protein in colon cancer tissue and infiltration site
　正常な大腸粘膜検体においては、ヒトＣ４．４Ａタンパク質は大腸上皮の底部において検出される場合があった。一方、癌組織検体においては、大腸癌である大半の場合（１２２例中９６例、７８．７％）において、ヒトＣ４．４Ａタンパク質の発現が検出された。癌病巣の表層部または中層部においては、ヒトＣ４．４Ａタンパク質の弱い発現がほとんどの場合は細胞質において検出され、一方浸潤先進部においては高頻度で細胞膜上にＣ４．４Ａタンパク質の発現が検出された（図４Ａ）。注目すべき事実として、膨大な数の癌組織検体のうちで、新生の間質に向かって面した細胞のみがヒトＣ４．４Ａタンパク質を発現していた（図４Ａ、左下パネル）。リンパ節または肺への転移においても、ヒトＣ４．４Ａタンパク質が浸潤周縁部において発現するという同様の発現パターンを示した。（図５） In normal colon mucosa samples, human C4.4A protein was sometimes detected at the bottom of the colon epithelium. On the other hand, expression of human C4.4A protein was detected in cancer tissue specimens in most cases of colon cancer (96/122, 78.7%). In the surface layer or middle layer of the cancer lesion, weak expression of human C4.4A protein is detected in the cytoplasm in most cases, whereas in the advanced invasion, the expression of C4.4A protein is detected on the cell membrane at a high frequency. (FIG. 4A). Of note, of the vast number of cancer tissue specimens, only the cells facing the neoplastic stroma expressed human C4.4A protein (FIG. 4A, lower left panel). In the metastasis to lymph nodes or lungs, a similar expression pattern was also shown in which the human C4.4A protein was expressed in the periphery of the infiltration. (Fig. 5)
　癌病巣の表層部または中層部における場合比較とした、癌浸潤先進部における染色パターンに基づいて、大腸癌の１２２症例を、次の４つのカテゴリに分類した：
（Ａ）　ヒトＣ４．４Ａタンパク質が癌浸潤先進部の細胞の細胞膜上に集積し、かつ癌浸潤先進部の細胞において癌病巣の表層部または中層部と比べて発現量が亢進している（ｎ＝３１）、
（Ｂ１）集積しているが、発現量の亢進なし（ｎ＝１１）、
（Ｂ２）集積していないが、発現量が亢進している（ｎ＝１４）、
（Ｃ）　集積、発現量の亢進のいずれもない（ｎ＝６６）。（表１）。 Based on the staining pattern in the advanced cancer invasion, compared to the case in the surface layer or middle layer of the cancer lesion, 122 cases of colorectal cancer were classified into the following four categories:
(A) Human C4.4A protein is accumulated on the cell membrane of cells in advanced cancer invasion, and the expression level is increased in cells in advanced cancer invasion compared to the surface layer or middle layer of cancer lesions (n = 31),
(B1) Accumulated but no increase in expression level (n = 11),
(B2) is not accumulated, but the expression level is increased (n = 14),
(C) Neither accumulation nor increased expression level (n = 66). (Table 1).
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
　Ａ型の発現パターンと、他の型（Ｂ１＋Ｂ２＋Ｃ）とにおいて、表２に列挙する種々の臨床的および病理学的パラメーターについて比較したところ、Ａ型はより高い腫瘍浸潤深度、リンパ節転移、および血行性転移（血管浸潤）と相関していた（それぞれについてＰ＝０．０４９、Ｐ＝０．０３６、Ｐ＝０．００２）。癌のステージがＩＩ期およびＩＩＩ期の患者群（ｎ＝８２）について性別、年齢、腫瘍サイズ、分化度、浸潤深度、リンパ節転移、および血行性転移（血管浸潤）について解析したところ、Ａ型は血行性転移（血管浸潤）とのみ相関していた（Ｐ＝０．００２７）。 Comparison of various clinical and pathological parameters listed in Table 2 between type A expression pattern and other types (B1 + B2 + C) showed that type A had higher tumor invasion depth, lymph node metastasis, and circulation It was correlated with sexual metastasis (vascular invasion) (P = 0.049, P = 0.036, P = 0.002 for each). A group of patients whose stage of cancer was stage II and stage III (n = 82) was analyzed for sex, age, tumor size, degree of differentiation, depth of invasion, lymph node metastasis, and hematogenous metastasis (vascular invasion). Correlated only with hematogenous metastases (vascular invasion) (P = 0.527).
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002
　２．腫瘍蔟出とヒトＣ４．４Ａタンパク質の発現パターンとの関係
　腫瘍蔟出は、腫瘍の悪性浸潤性の指標であり、腫瘍蔟出が高頻度に観察される場合（蔟出点数≧１０）は、腫瘍蔟出が低頻度である場合（蔟出点数０～９）と比べて、顕著に予後が不良である（全生存率についてはＰ＝０．００２［ステージＩ期～ＩＶ期、ｎ＝１２２］、無病生存率についてはＰ＝０．００４［ステージＩＩ期およびＩＩＩ期、ｎ＝８２］）ことは前出の報告の通りである。免疫染色を行ったところ、蔟出細胞においてヒトＣ４．４Ａが細胞膜上で濃く染色されることが高頻度で観察された（図４Ｂ）。蔟出頻度と浸潤先進部におけるヒトＣ４．４Ａの発現パターンの相関関係を調べたところ、２つの因子の間で顕著な相関関係が認められた（Ｐ＜０．０００１、図４Ｃ、表３）。 2. Relationship between tumor exudation and human C4.4A protein expression pattern Tumor exudation is an indicator of tumor malignant invasiveness, and when tumor exudation is observed frequently (number of exudation points ≧ 10), The prognosis is significantly worse than when the tumor eruption is infrequent (extraction score 0-9) (P = 0.002 [Stage I-IV, n = 122 for overall survival) As for the disease-free survival rate, P = 0.004 [Stage II and III, n = 82]), as previously reported. When immunostaining was performed, it was frequently observed that human C4.4A was stained deeply on the cell membrane in the shed cells (FIG. 4B). When the correlation between the frequency of sputum and the expression pattern of human C4.4A in the advanced infiltrating region was examined, a significant correlation was found between the two factors (P <0.0001, FIG. 4C, Table 3). .
Figure JPOXMLDOC01-appb-T000003
Figure JPOXMLDOC01-appb-T000003
　３．生存率解析
　前述の大阪大学外科学講座における手術時に、大腸癌患者より採取した１２２症例のヒトＣ４．４Ａタンパク質の局在の変化（Ａ＋Ｂ１）または発現強度の変化（Ａ＋Ｂ２）のいずれとも、より短い５年間全生存率（ＯＳ）と相関する（Ｐ＝０．０１４およびＰ＝０．００６、図６）ことを、生存率解析は示している。より顕著な差が、Ａ型と他の型（Ｂ１＋Ｂ２＋Ｃ）の間で認められた（Ｐ＝０．０００９、図６）。Ｂ１型およびＢ２型は、Ｃ型との間に顕著な差は認められなかった（図６）。癌のステージがＩＩ期およびＩＩＩ期の患者群について、外科治療後の５年間無病生存率（ＤＦＳ）について解析したところ、同様の結果が得られた（図７）。 3. Survival analysis At the time of surgery in the aforementioned Osaka University Surgery Course, 122 cases of changes in the localization of human C4.4A protein (A + B1) or change in expression intensity (A + B2) in 122 cases collected from colon cancer patients are shorter. Survival analysis shows that it correlates with 5-year overall survival (OS) (P = 0.014 and P = 0.006, FIG. 6). A more significant difference was observed between type A and other types (B1 + B2 + C) (P = 0.0009, FIG. 6). The B1 type and the B2 type were not significantly different from the C type (FIG. 6). Similar results were obtained when patients with stage II and stage III cancer were analyzed for disease-free survival (DFS) for 5 years after surgery (FIG. 7).
　一変量解析の結果は、腫瘍サイズが大きいこと、ならびにリンパ節転移、遠隔転移、および腹膜播種の存在、ならびに高頻度蔟出が、ヒトＣ４．４Ａタンパク質のＡ型発現パターンに加えて、不良な５年間ＯＳの前兆であることを示した。最も顕著な予後因子を判定するために多変量解析を行ったところ、ヒトＣ４．４Ａタンパク質のＡ型発現パターン、腫瘍サイズが大きいこと、リンパ節転移、遠隔転移、および腹膜転移は独立した予後因子であると同定されたが、高頻度蔟出は保持されなかった（表４、５）。癌のステージがＩＩ期およびＩＩＩ期の患者群（ｎ＝８２）において、一変量解析によるヒトとＣ４．４Ａタンパク質のＡ型発現パターン、高頻度蔟出、およびリンパ節転移の存在が短いＤＦＳの前兆であった。多変量解析の結果によると、ヒトＣ４．４Ａタンパク質のＡ型発現パターンのみが独立した予後因子であることが保持された（表６、７）。 The results of univariate analysis show that the large tumor size and the presence of lymph node metastasis, distant metastasis, and peritoneal dissemination, and frequent exudation are poor in addition to the type A expression pattern of human C4.4A protein It was a precursor to OS for 5 years. Multivariate analysis was performed to determine the most prominent prognostic factors, and human C4.4A protein type A expression pattern, large tumor size, lymph node metastasis, distant metastasis, and peritoneal metastasis were independent prognostic factors However, high-frequency sputum was not retained (Tables 4 and 5). In patients with stage II and stage III cancer (n = 82), univariate analysis of human and C4.4A protein A-type expression patterns, frequent eruption, and the presence of DFS with short lymph node metastases It was a precursor. According to the results of multivariate analysis, it was retained that only the type A expression pattern of human C4.4A protein was an independent prognostic factor (Tables 6 and 7).
Figure JPOXMLDOC01-appb-T000004
Figure JPOXMLDOC01-appb-T000004
Figure JPOXMLDOC01-appb-T000005
Figure JPOXMLDOC01-appb-T000005
Figure JPOXMLDOC01-appb-T000006
Figure JPOXMLDOC01-appb-T000006
Figure JPOXMLDOC01-appb-T000007
Figure JPOXMLDOC01-appb-T000007
　＜ＩＩＩ．考察＞
　このように、大腸癌患者の予後観察を行ったところ、癌組織献体においてヒトＣ４．４Ａタンパク質のＡ型発現パターンが見られた患者においては、統計学的に有意に予後不良であった。すなわち、ヒトＣ４．４Ａタンパク質のＡ型発現パターンを予後不良の判定基準として用いることができることがここに示されている。 <III. Discussion>
Thus, when the prognosis observation of the colon cancer patient was performed, the prognosis was statistically significantly poor in the patient in which the type A expression pattern of the human C4.4A protein was seen in the cancer tissue donation. That is, it is shown here that the type A expression pattern of human C4.4A protein can be used as a criterion for poor prognosis.
　［実施例３］
　標的ペプチドと染色パターンとの関係の検証
　＜Ｉ．実験材料と実験方法＞
　実施例１と同様にして、配列番号１に示すアミノ酸配列のペプチドを標的ペプチドとして、抗Ｃ４．４Ａ－２抗体とは異なる２羽のウサギから、ポリクローナル抗体を得た。それぞれ、抗Ｃ４．４Ａ－２－２ポリクローナル抗体および抗Ｃ４．４Ａ－２－３ポリクローナル抗体と命名した。 [Example 3]
Verification of relationship between target peptide and staining pattern <I. Experimental materials and methods>
In the same manner as in Example 1, a polyclonal antibody was obtained from two rabbits different from the anti-C4.4A-2 antibody using the peptide having the amino acid sequence shown in SEQ ID NO: 1 as the target peptide. They were named anti-C4.4A-2-2 polyclonal antibody and anti-C4.4A-2-3 polyclonal antibody, respectively.
　また、ヒトＣ４．４Ａタンパク質の別の部位である、配列番号４に示すアミノ酸配列（Ｃ４．４Ａ（１１９）ペプチド：ヒトＣ４．４Ａタンパク質の１１９～１４０番目のアミノ酸配列に相当）のペプチド、および配列番号５に示すアミノ酸配列（Ｃ４．４Ａ（２７７）ペプチド：ヒトＣ４．４Ａタンパク質の２７７～２９７番目のアミノ酸配列に相当）のペプチドを標的ペプチドとしたポリクローナル抗体を得た。それぞれ、抗Ｃ４．４Ａ（１１９）ポリクローナル抗体および抗Ｃ４．４Ａ（２７７）ポリクローナル抗体と命名した。図８に、ヒトＣ４．４Ａタンパク質における各々の標的ペプチドの部位を示す。 A peptide having the amino acid sequence shown in SEQ ID NO: 4, which is another site of the human C4.4A protein (C4.4A (119) peptide: corresponding to amino acids 119 to 140 of the human C4.4A protein), and A polyclonal antibody was obtained using the peptide having the amino acid sequence shown in SEQ ID NO: 5 (C4.4A (277) peptide: corresponding to the 277-297th amino acid sequence of human C4.4A protein) as the target peptide. They were named anti-C4.4A (119) polyclonal antibody and anti-C4.4A (277) polyclonal antibody, respectively. FIG. 8 shows the site of each target peptide in the human C4.4A protein.
　得られた各々の抗体について、大腸癌患者由来の試料における免疫組織化学による染色パターンを、実施例１で取得した抗Ｃ４．４Ａ－２抗体との染色パターンと比較した。免疫組織化学は、実施例１および２と同様にして行なった。 For each of the antibodies obtained, the staining pattern by immunohistochemistry in a sample derived from a colon cancer patient was compared with the staining pattern with the anti-C4.4A-2 antibody obtained in Example 1. Immunohistochemistry was performed as in Examples 1 and 2.
　＜ＩＩ．実験結果と考察＞
　図９に示すように、抗Ｃ４．４Ａ－２－２ポリクローナル抗体および抗Ｃ４．４Ａ－２－３ポリクローナル抗体については、浸潤先進部においては細胞膜上に染色が観察された。これは、抗Ｃ４．４Ａ－２ポリクローナル抗体と同様の染色パターンであった。 <II. Experimental results and discussion>
As shown in FIG. 9, for the anti-C4.4A-2-2 polyclonal antibody and the anti-C4.4A-2-3 polyclonal antibody, staining was observed on the cell membrane at the advanced infiltration site. This was a staining pattern similar to that of the anti-C4.4A-2 polyclonal antibody.
　一方、図１０に示すように、抗Ｃ４．４Ａ（１１９）ポリクローナル抗体および抗Ｃ４．４Ａ（２７７）ポリクローナル抗体を用いた場合は、癌浸潤先進部における特徴的な染色パターンは、観察されなかった。 On the other hand, as shown in FIG. 10, when the anti-C4.4A (119) polyclonal antibody and the anti-C4.4A (277) polyclonal antibody were used, a characteristic staining pattern in the advanced part of cancer invasion was not observed. .
　以上の結果は、実施例２で観察された癌浸潤先進部における染色パターンは、ヒトＣ４．４Ａタンパク質の特定の部位を標的とする抗体において、特異的に観察されることを示している。 The above results indicate that the staining pattern in the cancer invasion advanced part observed in Example 2 is specifically observed in the antibody targeting a specific site of human C4.4A protein.
　［実施例４］
モノクローナル抗体の作製と特異性の検証
＜Ｉ．実験材料と実験方法＞
　１．モノクローナル抗体の作製
　下記の手順に従い、モノクローナル抗体を産生するハイブリドーマを得た。
[1]標的ペプチドとして、配列番号１に示すアミノ酸配列を有するペプチドのN末端にシステインと１残基付した配列を有するペプチドを合成した。
[2]上記ペプチドをウシサイログロブリンへ結合し、これをマウスへ免疫した。
免疫動物は、以下の通りであるSPF/VAF B6D2F1/Crlj (BDF1マウス)雄、6週齢（日本チャールスリバーより取得）を2匹、およびSPF/VAF BALB/cAnNCrlCrlj (BALB/cマウス)雄、6週齢（日本チャールスリバーより取得）を2匹、である。
免疫条件は、50μg/匹/回 (初回FCA, 2回目以降FICAのエマルジョン)を腹部と背部の皮下および皮内へ7日毎に1回、計4回の免疫として行なった。
[3]試験採血を抗原固相ELISA及び免疫組織染色で評価し、抗体力価の亢進が見られた個体を選抜した。
[4]選抜したマウス個体から脾臓細胞およびリンパ節を調製し、ミエローマ（P3X63Ag8.653）と融合し、融合細胞（ハイブリドーマ）を得た。
[5]得られた融合細胞を限界希釈法による細胞播種を行った。ついで、培養上清を、標的ペプチドを固定した抗原固相プレートに対してELISA法を行い、標的力価に対する抗原力価が高い抗体を産生するハイブリドーマを選択した。 [Example 4]
Preparation of monoclonal antibody and verification of specificity <I. Experimental materials and methods>
1. Preparation of monoclonal antibody A hybridoma producing a monoclonal antibody was obtained according to the following procedure.
[1] As a target peptide, a peptide having a sequence with one residue of cysteine added to the N-terminus of the peptide having the amino acid sequence shown in SEQ ID NO: 1 was synthesized.
[2] The peptide was conjugated to bovine thyroglobulin and immunized to mice.
The immunized animals are: SPF / VAF B6D2F1 / Crlj (BDF1 mouse) male, 2 6-week-old (acquired from Charles River Japan), and SPF / VAF BALB / cAnNCrlCrlj (BALB / c mouse) male, Two, 6 weeks old (obtained from Japan Charles River).
The immunization conditions were 50 μg / animal / dose (initial FCA, second and subsequent FICA emulsions) subcutaneously and intradermally in the abdomen and back once every 7 days for a total of 4 immunizations.
[3] Test blood collection was evaluated by antigen solid-phase ELISA and immunohistochemical staining, and individuals with increased antibody titers were selected.
[4] Spleen cells and lymph nodes were prepared from the selected mouse individuals and fused with myeloma (P3X63Ag8.653) to obtain fused cells (hybridoma).
[5] The obtained fused cells were seeded by limiting dilution. Subsequently, the culture supernatant was subjected to ELISA on an antigen solid phase plate on which the target peptide was immobilized, and hybridomas producing an antibody having a high antigen titer relative to the target titer were selected.
　得られたハイブリドーマの一つを11A1と命名し、以下の解析に用いた。 One of the obtained hybridomas was named 11A1 and used for the following analysis.
　なお、ハイブリドーマの作製およびモノクローナル抗体の精製は、株式会社免疫生物研究所に委託して行なった。 The production of hybridomas and the purification of monoclonal antibodies were outsourced to the Institute for Immunobiology.
　作製したハイブリドーマ11A1は、２０１１年４月２７日（原寄託日）に、日本国日本国千葉県木更津市かずさ鎌足２－５－８に住所を有する独立行政法人製品評価技術基盤機構特許微生物寄託センターに受託され、２０１１年５月３１日に原寄託よりブダペスト条約に基づく寄託への移管請求が受託された。その受託番号は、NITE BP-1090であり、受託者が付した識別のための表示は「CA4.4A Hybridoma 11A1」である。 The produced hybridoma 11A1 was deposited on April 27, 2011 (original deposit date), which was deposited with the Patent Microorganisms of the National Institute of Technology and Evaluation of the National Institute of Technology and Evaluation, addressed 2-5-8, Kazusa Kamashita, Kisarazu, Chiba, Japan. On May 31, 2011, a request for transfer from the original deposit to a deposit based on the Budapest Treaty was accepted on May 31, 2011. The accession number is NITE BP-1090, and the display for identification given by the trustee is “CA4.4A Hybridoma 11A1”.
　２．免疫組織化学
　上記で得たハイブリドーマ11A1が産生するモノクローナル抗体（以下、抗Ｃ４．４Ａ－２モノクローナル抗体と記載する。）を用いて、大腸癌患者由来の試料における免疫組織化学による染色パターンを、実施例１で取得した抗Ｃ４．４Ａ－２抗体との染色パターンと比較した。 2. Immunohistochemistry Using the monoclonal antibody produced by the hybridoma 11A1 obtained above (hereinafter referred to as anti-C4.4A-2 monoclonal antibody), a staining pattern by immunohistochemistry was performed on a sample derived from a colon cancer patient. The staining pattern was compared with the anti-C4.4A-2 antibody obtained in Example 1.
　また、下記の手法により、過剰量のヒトＣ４．４Ａ－２抗原ペプチドにあらかじめ吸着させた抗Ｃ４．４Ａ－２モノクローナル抗体を用いた染色パターンも得た。
[1]抗体希釈バッファー（1 % BSA in PBS）へ抗体およびペプチドを、抗体:ペプチドが1 mol : 20 molとなるように添加。対照として、ペプチドに代えて精製水を添加したものを用いる。
[2]4℃で一晩、転倒混和。 In addition, a staining pattern using an anti-C4.4A-2 monoclonal antibody previously adsorbed to an excessive amount of human C4.4A-2 antigenic peptide was also obtained by the following method.
[1] Add antibody and peptide to antibody dilution buffer (1% BSA in PBS) so that antibody: peptide is 1 mol: 20 mol. As a control, a solution in which purified water is added instead of the peptide is used.
[2] Inverted overnight at 4 ° C.
　３．ウェスタンブロット法
　実施例１と同様にして、[1]抗Ｃ４．４Ａ－２ポリクローナル抗体、[2]過剰量のヒトＣ４．４Ａ－２抗原ペプチドにあらかじめ吸着させた抗Ｃ４．４Ａ－２ポリクローナル抗体、[3]抗Ｃ４．４Ａ－２モノクローナル抗体、および[4]過剰量のヒトＣ４．４Ａ－２抗原ペプチドにあらかじめ吸着させた抗Ｃ４．４Ａ－２モノクローナル抗体を用いて、ウェスタンブロットを行なった。試料として、大腸癌由来細胞株ＨＣＴ１１６を用いた。 3. Western blotting In the same manner as in Example 1, [1] anti-C4.4A-2 polyclonal antibody, [2] anti-C4.4A-2 polyclonal antibody previously adsorbed to an excess amount of human C4.4A-2 antigenic peptide Western blot was performed using [3] anti-C4.4A-2 monoclonal antibody and [4] anti-C4.4A-2 monoclonal antibody previously adsorbed to an excess amount of human C4.4A-2 antigenic peptide. . As a sample, a colon cancer-derived cell line HCT116 was used.
　＜ＩＩ．実験結果＞
　図１１に、大腸癌患者由来の試料の連続切片における、抗Ｃ４．４Ａ－２モノクローナル抗体と抗Ｃ４．４Ａ－２ポリクローナル抗体の染色パターンを示す。抗Ｃ４．４Ａ－２モノクローナル抗体により得られる染色パターンは、抗Ｃ４．４Ａ－２ポリクローナル抗体の場合と同様に、癌浸潤先進部においては高頻度で細胞膜上に染色が観察された。 <II. Experimental results>
FIG. 11 shows staining patterns of anti-C4.4A-2 monoclonal antibody and anti-C4.4A-2 polyclonal antibody in serial sections of a sample derived from a colon cancer patient. As in the case of the anti-C4.4A-2 polyclonal antibody, the staining pattern obtained with the anti-C4.4A-2 monoclonal antibody was frequently observed on the cell membrane in the cancer invasion advanced portion.
　図１２に、過剰量のヒトＣ４．４Ａ－２抗原ペプチドにあらかじめ吸着させた抗Ｃ４．４Ａ－２モノクローナル抗体を用いた染色パターンを示す。癌浸潤先進部における特異的な染色パターンが、観察されなかった。 FIG. 12 shows a staining pattern using an anti-C4.4A-2 monoclonal antibody previously adsorbed to an excessive amount of human C4.4A-2 antigenic peptide. No specific staining pattern was observed in advanced cancer invasion.
　図１３に、ウェスタンブロット解析の結果を示す。抗Ｃ４．４Ａ－２モノクローナル抗体を用いて、抗Ｃ４．４Ａ－２ポリクローナル抗体と同様に約４０ｋＤａの位置にバンド（図中、矢印で指し示すバンド）を検出することができた。また、約４０ｋＤａの位置のバンドは、過剰量のヒトＣ４．４Ａ－２抗原ペプチドにあらかじめ吸着させた抗Ｃ４．４Ａ－２ポリクローナル抗体および過剰量のヒトＣ４．４Ａ－２抗原ペプチドにあらかじめ吸着させた抗Ｃ４．４Ａ－２モノクローナル抗体を用いた場合においては、検出されなかった。 FIG. 13 shows the results of Western blot analysis. Using the anti-C4.4A-2 monoclonal antibody, a band (a band indicated by an arrow in the figure) could be detected at a position of about 40 kDa as in the case of the anti-C4.4A-2 polyclonal antibody. In addition, a band at a position of about 40 kDa is preliminarily adsorbed to an anti-C4.4A-2 polyclonal antibody previously adsorbed to an excess amount of human C4.4A-2 antigen peptide and to an excess amount of human C4.4A-2 antigen peptide. When anti-C4.4A-2 monoclonal antibody was used, it was not detected.
　＜ＩＩＩ．考察＞
　以上の結果は、CA4.4A Hybridoma 11A1が産生するモノクローナル抗体が、配列番号１に示すアミノ酸配列のペプチドを特異的に認識する抗体であり、免疫組織化学およびウェスタンブロット解析において実施例１で得た抗Ｃ４．４Ａ－２ポリクローナル抗体と同様の反応性を有する抗体である。すなわち、CA4.4A Hybridoma 11A1が産生するモノクローナル抗体は、本発明の予後判定方法において好適に使用することができる。 <III. Discussion>
The above results show that the monoclonal antibody produced by CA4.4A Hybridoma 11A1 specifically recognizes the peptide having the amino acid sequence shown in SEQ ID NO: 1, and was obtained in Example 1 in immunohistochemistry and Western blot analysis. This antibody has the same reactivity as the anti-C4.4A-2 polyclonal antibody. That is, the monoclonal antibody produced by CA4.4A Hybridoma 11A1 can be suitably used in the prognosis determination method of the present invention.
　［実施例５］
ヒトＣ４．４Ａタンパク質の発現パターンと、リンパ節転移および血行性転移との相関
　ヒトＣ４．４Ａタンパク質の発現パターンと、リンパ節転移および血行性転移との相関をより詳細に検証するために、実施例３で用いた１２２症例の大腸癌組織検体に加えて、遠隔転移を生じた１０症例（大阪大学外科学講座）の大腸癌組織検体を加えた計１３２症例について、実施例２と同様に統計解析をおこなった。統計解析の結果を、表８～１３に示す。 [Example 5]
Correlation between human C4.4A protein expression pattern and lymph node metastasis and hematogenous metastasis To verify in more detail the correlation between human C4.4A protein expression pattern and lymph node metastasis and hematogenous metastasis In addition to 122 cases of colorectal cancer tissue specimens used in Example 3, a total of 132 cases including 10 cases (Osaka University Surgery Department) colon cancer tissue specimens with distant metastases were added as in Example 2. Analysis was performed. The results of statistical analysis are shown in Tables 8-13.
Figure JPOXMLDOC01-appb-T000008
Figure JPOXMLDOC01-appb-T000008
Figure JPOXMLDOC01-appb-T000009
Figure JPOXMLDOC01-appb-T000009
Figure JPOXMLDOC01-appb-T000010
Figure JPOXMLDOC01-appb-T000010
Figure JPOXMLDOC01-appb-T000011
Figure JPOXMLDOC01-appb-T000011
Figure JPOXMLDOC01-appb-T000012
Figure JPOXMLDOC01-appb-T000012
Figure JPOXMLDOC01-appb-T000013
Figure JPOXMLDOC01-appb-T000013
　特に着目すべき点として、遠隔転移を生じた１０症例（大阪大学外科学講座）を加えた症例群の解析からは、ヒトＣ４．４Ａタンパク質のＡ型発現パターンとリンパ節転移とは相関しないことが明らかとなった（表９、Ｐ値＝0.133）。一方で、ヒトＣ４．４Ａタンパク質のＡ型発現パターンと血行性転移との相関は保持された（表９、Ｐ値＝0.003）。 Of particular note is that there is no correlation between the type A expression pattern of human C4.4A protein and lymph node metastasis from the analysis of the case group including 10 cases (Osaka University Department of Surgery) with distant metastasis. Became clear (Table 9, P value = 0.133). On the other hand, the correlation between the type A expression pattern of human C4.4A protein and hematogenous metastasis was maintained (Table 9, P value = 0.003).
　＜総合考察＞
　以上の実施例により、癌組織献体においてヒトＣ４．４Ａタンパク質のＡ型発現パターンが見られた患者においては、統計学的に有意に予後不良であった（実施例２）。また、詳細な解析の結果、ヒトＣ４．４Ａタンパク質のＡ型発現パターンは、血行性転移とは相関するものの、リンパ節転移とは相関が小さいことが示唆された（実施例５）。また、ヒトＣ４．４Ａタンパク質のＡ型発現パターンは、リンパ節転移と比べて、癌再発のリスクをより顕著に示す因子であることも示唆された。 <General consideration>
According to the above Examples, the prognosis was statistically significantly poor in patients whose cancer tissue donation showed type A expression pattern of human C4.4A protein (Example 2). Further, as a result of detailed analysis, it was suggested that the type A expression pattern of human C4.4A protein correlates with hematogenous metastasis but has little correlation with lymph node metastasis (Example 5). It was also suggested that the type A expression pattern of the human C4.4A protein is a factor that more significantly shows the risk of cancer recurrence than lymph node metastasis.
　これらの結果は、ヒトＣ４．４Ａタンパク質のＡ型発現パターンを予後不良の判定基準として用いることを示しており、さらには、ヒトＣ４．４Ａタンパク質のＡ型発現パターンは血行性転移に起因する癌再発リスクの指標であることを示している。 These results indicate that the type A expression pattern of human C4.4A protein is used as a criterion for poor prognosis, and further, the type A expression pattern of human C4.4A protein is a cancer caused by hematogenous metastasis. It is an indicator of recurrence risk.
　従来、リンパ節転移の有無が、外科手術後に抗癌剤投与などの癌再発を予防するためのアフターケアを行なうか否かの目安となっている。リンパ性転移陰性の場合は、予防的アフターケアを行なわないことが一般的であるが、リンパ性転移陰性において１０～２０％程度の患者において癌が再発するに過ぎない。一方、リンパ性転移陽性の場合は、抗癌剤投与などの予防的アフターケアを行なっているものの、リンパ性転移陽性において癌が再発する割合は２５～４０％程度である。これは、リンパ節転移の有無を指標とした場合には、本来予防的アフターケアを必要としない患者に、身体的、精神的および経済的な負担を強いていることを意味する。 Conventionally, the presence or absence of lymph node metastasis has become an indication of whether or not to perform aftercare to prevent cancer recurrence such as administration of anticancer agents after surgery. When lymphatic metastasis is negative, prophylactic aftercare is generally not performed, but cancer is recurrent only in about 10 to 20% of patients with lymphatic metastasis negative. On the other hand, when lymphatic metastasis is positive, preventive aftercare such as administration of an anticancer agent is performed, but the rate of cancer recurrence when lymphatic metastasis is positive is about 25 to 40%. This means that when the presence or absence of lymph node metastasis is used as an indicator, patients who do not need preventive aftercare are physically, mentally and economically burdened.
　本発明の抗体を用いた方法は、ヒトＣ４．４Ａタンパク質のＡ型発現パターンにより、血行性転移のリスクを検出できると考えられる。そのため、従来のリンパ節転移の有無を指標とした場合には検出することができなかった、血行性転移に起因する癌再発を予見できると考えられる。 It is considered that the method using the antibody of the present invention can detect the risk of hematogenous metastasis from the type A expression pattern of human C4.4A protein. Therefore, it is considered that cancer recurrence due to hematogenous metastasis, which could not be detected when using the presence or absence of conventional lymph node metastasis as an index, can be predicted.
　本発明の抗体を用いた方法により、癌手術後に癌再発のリスクが高い患者を、高い精度で予期することができる。すなわち、予防的アフターケアが必要でない患者においてはアフターケアを行なわず、真に予防的アフターケアを必要とする患者において重点的にアフターケアを行うことが容易となる。 By the method using the antibody of the present invention, a patient who has a high risk of cancer recurrence after cancer surgery can be predicted with high accuracy. That is, aftercare is not performed in patients who do not require preventive aftercare, and it becomes easy to focus on aftercare in patients who truly need preventive aftercare.

Claims (15)

  1. 　配列番号１に示すアミノ酸配列の領域に特異的に結合する抗ヒトＣ４．４Ａタンパク質抗体。 An anti-human C4.4A protein antibody that specifically binds to the region of the amino acid sequence shown in SEQ ID NO: 1.
  2. 　ポリクローナル抗体である、請求項１に記載の抗体。 The antibody according to claim 1, which is a polyclonal antibody.
  3. 　モノクローナル抗体である、請求項１に記載の抗体 The antibody according to claim 1, which is a monoclonal antibody.
  4. 　受託番号：NITE BP-1090で特定されるハイブリドーマが産生するモノクローナル抗体である、請求項３に記載のモノクローナル抗体。 The monoclonal antibody according to claim 3, which is a monoclonal antibody produced by a hybridoma identified by accession number: NITENBP-1090.
  5. 　癌患者の予後を判定する方法であって、
    （１）癌患者から採取した癌組織において、請求項１から４のいずれかに記載の抗体でヒトＣ４．４Ａタンパク質の発現を検出する工程、
    （２）前記工程（１）の検出結果に基づいて、癌浸潤先進部の細胞において、細胞膜と細胞質との間でヒトＣ４．４Ａタンパク質の局在を比較し、かつ癌浸潤先進部と癌病巣の表層部または中層部との間で、ヒトＣ４．４Ａタンパク質の発現量を比較する工程、および
    （３）前記工程（２）の比較に基づいて、前記ヒトＣ４．４Ａタンパク質が前記癌浸潤先進部の細胞の細胞膜上に集積し、かつ前記ヒトＣ４．４Ａタンパク質の発現量が前記癌浸潤先進部の細胞において、前記癌病巣の表層部または中層部と比べて亢進している場合は、予後不良と判定する工程、を含む方法。     A method for determining the prognosis of a cancer patient,
    (1) a step of detecting expression of human C4.4A protein with the antibody according to any one of claims 1 to 4 in a cancer tissue collected from a cancer patient;
    (2) Based on the detection result of the step (1), the localization of the human C4.4A protein is compared between the cell membrane and the cytoplasm in the cells of the advanced cancer invasion, and the advanced cancer invasion and the cancer lesion are compared. Comparing the expression level of the human C4.4A protein with the surface layer portion or the middle layer portion of (3), and (3) based on the comparison in the step (2), the human C4.4A protein is Prognosis when the expression level of the human C4.4A protein is accumulated on the cell membrane of some cells and is increased in the cells in the cancer invasion advanced portion as compared with the surface layer portion or middle layer portion of the cancer lesion. Determining a failure.
  6. 　予後が、癌再発リスクに基づく予後である、請求項５に記載の方法。 6. The method according to claim 5, wherein the prognosis is a prognosis based on a risk of cancer recurrence.
  7. 　癌再発が、血行性転移に起因する癌再発である、請求項６に記載の方法。 The method according to claim 6, wherein the cancer recurrence is cancer recurrence caused by hematogenous metastasis.
  8. 　癌患者が消化器系の癌の患者である請求項５～７のいずれか１項に記載の方法。 The method according to any one of claims 5 to 7, wherein the cancer patient is a gastrointestinal cancer patient.
  9. 　癌患者の予後を判定するためのキットであって、請求項１から４のいずれかに記載の抗体を含んでなるキット。 A kit for determining the prognosis of a cancer patient, comprising the antibody according to any one of claims 1 to 4.
  10. 　予後が、癌再発リスクに基づく予後である、請求項９に記載のキット。 The kit according to claim 9, wherein the prognosis is a prognosis based on a cancer recurrence risk.
  11. 　癌再発が、血行性転移に起因する癌再発である、請求項１０に記載のキット。 The kit according to claim 10, wherein the cancer recurrence is cancer recurrence caused by hematogenous metastasis.
  12. 　癌患者が消化器系の癌の患者である、請求項９～１１のいずれか１項に記載のキット。 The kit according to any one of claims 9 to 11, wherein the cancer patient is a gastrointestinal cancer patient.
  13. 　請求項１から４のいずれかに記載の抗体を含む、癌再発リスクの診断試薬。 A diagnostic reagent for cancer recurrence risk, comprising the antibody according to any one of claims 1 to 4.
  14. 　癌再発が、血行性転移に起因する癌再発である、請求項１３に記載の診断試薬。 The diagnostic reagent according to claim 13, wherein the cancer recurrence is cancer recurrence caused by hematogenous metastasis.
  15. 　癌が消化器系の癌の患である、請求項１３または１４に記載の診断試薬。
          The diagnostic reagent according to claim 13 or 14, wherein the cancer is a disease of a cancer of the digestive system.
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